CA3170375A1

CA3170375A1 - Engineered influenza neuraminidase antigens

Info

Publication number: CA3170375A1
Application number: CA3170375A
Authority: CA
Inventors: Neil P. King; Daniel Ellis; Masaru Kanekiyo; Julia LEDERHOFER; Barney S. Graham
Original assignee: Individual
Current assignee: University of Washington; US Department of Health and Human Services
Priority date: 2020-03-06
Filing date: 2021-03-04
Publication date: 2021-09-10
Also published as: WO2021178621A1; JP2023521946A; KR20220155313A; AU2021231832A1; EP4114447A1; US20230090779A1; CN115461074A

Abstract

The disclosure provides non-naturally occurring mutant neuraminidase (NA) polypeptides that improve expression and/or modifies the open/closed tetramerie conformational state of the NA polypeptide, and uses thereof.

Description

Engineered influenza neuraminidase antigens Cross Reference This application claims priority to U.S. Provisional Patent Application Serial No.
62/986295 filed March 6, 2020, incorporated by reference herein in its entirety.
.10 Background Tufluenz.a viruses claim countless lives and pose a significant public health and economic burden globally every year. The development of highly effective vaccines to ever-evolving influenza viruses has been a major public health priority. There are two Major viral glycoproteins on the influenza virion, the hemagglutinin (HA) and the neuraminidase (NA), which facilitate viral entry.and egress from host cells, respectively. NA is a homotetrameric, type II integral membrane protein, the N terminus of which is oriented towards the interior of the virus. The C-terminal globular head domain of NA contains six.
topologically identical beta sheets arranged in a propeller-like structure, comprises the discontinuous catalytic site, and is supported by a stalk domain. NA cleaves terminal sialic acid from glycans on the host.
cell surface, thereby releasing nascent viral particles. Additionally, it is believed that NA
might play a role in viral entry. These characteristics make NA an attractive target as a vaccine immunogen, yet NA has historically been neglected in vaccine development.
Summary In one aspect, the disclosure provides non-naturally occurring mutant neuraminidase (NA) polypeptides that improve expression and/or modifies the open/closed tetrameric conformational state of the NA polypeptide. In one embodiment, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%., 98%, or 99% identical to the NI .NA polypeptide of SEQ ID NO: 1, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, or all 7 of the fbilowing amino acid residues relative to SEQ ID NO:1 when aligned by protocol I or protocol 2:
1611'/A, 105A, 165171, 166P, 196Q, 203Y, 444V; or (h) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the NI NA polypepticle of SEQ NO:1, and wherein the non-naturally occurring .polypeptide includes 2, 3.4, 5, 6, 7, 8, 9, 1.0, 11, 12, 13, 14, 15, 16, or all 17 of the following amino acid residues relative to SEQ ID NO:1 whenaligned by protocol 1 or protocol 2:
161T/A1WS/T, 10014 408M, 419V, 99P, 1.03.N, 105A, 131Q/M, 1631/L, 165T/SN/A/1, 166P, 1771, 196Q/T, 203Y, 2051,4421. 444V.
In another embodiment, the polypeptide is (a) at least 75%, 80%, '85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypeptide of SEQ ID NO:2, and wherein the non-naturally occurring polypeptide includes 1;2, 3, 4,5, 6, 7, or all 8 of the following amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1-or protocol 2:
10IC, 13.1M, 162P, 165SIT, 166V, I95Q, 202Y, 443S; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypeptide of SEQ ID NO:2, And wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8. 9, 10, I I. or all 12 of the following amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol

2:
10ICIA, 103N, 105A, 106V, 131M, 162P, 1631, 1165S/T/A/I, 166V, 195Q, 202Y, 443S.
In another embodiment, the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N3 NA .polypeptide of SEQ ID NO:3, and wherein the non-naturally occurring poly-peptide includes 1, 2, 3,4. 5, 6, 7, 8, 9, 10, or all 11 of the following amino acid residues relative to SEQ ID NO:3 when aligned by protocol I or protocol 2:
103N, 105S, 131Q/M, 157T, 165S/1, 166P, 196Q, 203Y, 2051, 443S, 445V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N3 NA polypeptide of SEQ 'ID NO:3, and wherein the nOrtnaturaily occurring poly-peptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1.2, or all 13 of the following amino acid residues relative to SEQ ID NO:I when aligned by protocol I or protocol 2:
103N, 105S, I 06Võ 13 I 157T, 163L, 1655/1iV/A, 166P/V, 196Q, 203Y, 2051, 443S, 445V.
In a further embodiment, the polypeptide is (a) at least 75%, 80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N4 NA polypeptide of SEQ ID NO:4, and wherein the non-naturally occurring .polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following amino acid residues relative to SEQ ID NO:4 when aligned by protocol 1 or protocol 2:

160WSMA, 409M, 102N, 104S/A, 105V, 1120,130Q1M, 1621,õ 1645/T/All, 1651', 1761, 195Q, 202Y, 2041, 4431, 445V; or (b) at least 75%, 80%, 85%õ 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identical. to the N4 NA polypeptide of SEQ 11 NO:4, and wherein the non-naturally occurring polypeptide includes 2, 3,4, S. 6, 7, 8,9, I0, 11, 12, 13, 14, 15, or all 16 of the following amino- acid residues relative to SEQ ID NO:4 when aligned by protocol I or protocol 2:
tovismA, 409M, 102N, 104S/Aõ 105V, 1120, 130Q/M, 1621,, 164SIT/Af1, I65PN, 1761, 195Q, 202Y, 2041, 4431, 445V.
In one embodiment, the polypeptide is (a) at least 75%, 80%, :85%, 90%, 9.1%. 92%, 93%, 94%, 93%, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ NO:5, and wherein the non-naturally occurring polypeptide includes 1, 2, 3,4. 5, 6, 7, 8, 9, 10, 11, 12, or all 13 of the following amino acid residues relative to SEQ ID NO:5 when aligned by protocol I or protocol 2:
971,, 410M, 961', 98A, 100N, 102S/A, 1101), 1.28Q/M, 1601, 162V/A/1, 163V/P, 193Q/T, 4451; or (b) at least 75%, 80%, 85%, 90%. 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, or 99% identical to-the N5 NA .polypeptide of SEQ ED NO:5, and wherein the non-naturally occurring polypeptide, includes 2,3,4, 5, 6, 7, 8, 9, 10, 1.1, 12, 13, 14, 15, or all 16 of the following amino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2:
9713, 410M, 961'. 98A, .100N, 102S/A, 103V, 1.10D, 128Q/M, 154T, 1601, 162V/A/1/T, 1.63V/P, 1741, 1.93Q/T, 4451.
In a further embodiment, the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%., or 99% identical to the N6 NA pOlypeptide of SEQ ID NO:6, and Wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the following amino acid residues reiath's to SEQ ID NO:6 when aligned by protocol I. Or protocol 2:
99P, 103N, 113D, 131Q, 1611, 1621', 1631, 165SITIV/A, 196Q, 203Y, 445S; or (b) at least 75%, 80%, 85%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%. 98%, or 99% identical to the N6 NA polypeptide of SEQ ID NO:6, and wherein the non4tatural1y occurring .polypeptide includes 2, 3. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following amino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2:

3 99P, 103N, 1055, 106V, 113D, 131Q, 157T, 1611, 162P, 1631/L, 165S/T/V/A/I, 166V/P, 196Q, 203Y, 445S.
In one embodiment, the polypcptide is (a) at feast 75%, 80%, .85%, 90%, 91%, 92%, 93%, 94%õ 95%, 96%, 97%, 98%, or 99% identical to the N7 NA polypeptide of SEQ ID NO:7, and wherein the non-naturally occurring polypeptide includes 1,2, 3,4, 3, 6, 7, 8, 9, 10, or all 11 of the following amino acid residues relative to SEQ ID NO:7 when aligned by protocol 1 or protocol 2:
98P, 102N, 104S, 112D, 130Q, 156T, 1.621, 164 WAIL, 165V, 202Y, 448V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N7 NA .polypeptide of SEQ NO:7, and wherein the non-naturally occurring polypeptide includes 2, 3.4. 5, 6, 7, 8, 9, 10, II, 12, or all 13 of the following amino acid residues relative to SEQ ID NO:7 when aligned by protocol 1 or protocol 2:
9811, 102N, 104S, 1121), 130Q, 1561', 1611, 164V/A/I, 165V, 1761, 202Y, 4461, 448V.
In another embodiment, the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide of SEQ ID NO:8õ and wherein the non-naturally occurring polypeptide includes 1.2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the following amino acid residues relative to SEQ ID NO:8 when aligned by protocol 1 or protocol 2:
408M, LOIN, 103.A/S, 1 1 1 0, 129Q, 16IP/L, 1.63SITNIA/1, 164V, 194Q, 201Y, 4421; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 3, 9, 10, 11, 12, 13, 14. or all 15 of the following amino acid residues relative to SEQ ID NO:8 when aligned by protocol 1 or protocol 2:
98L, 408M, 101N, 103A/S, 104V, 111 0, 129Q/M, 161P/L, 163S/TN/A/I/S/T, 164V/P, 1751, 194Q, 201Y, 2031, 4421 In one embodiment, the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide includes 1,.2, 3, 4, 5,6, 7, 8, 9, or all 10 of the following amino acid residues relative to SEQ ID NO:9 when aligned by protocol Lor protocol 2:
94P, 95L. 1005, 126Q/M, 160V/A/I, 161V, 191Q, 198Y, 4395, 441V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally

4 occurring .polypeptide includes 2, 3,4, 5, 6, 7, 8, 9, 1.0, 11, 12, 13, or all 14 of the following amino acid residues relative: to SEQ ID NO:9 when aligned by protocol I or protocol 2:
94P, 95L, 98N, .100S/A, 126Q/14, 152T, 1581, 160VIAILIT, 161V, 191Q, 198'Y, 2001, 439S, .441V.
In one embodiment, the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ
ID
NO:9, and wherein the non-naturally occurring polypeptide includes a 160Q
amino acid mutation relative to SEQ ID NO:9 when aligned by protocol I , or may include a combination of 160Q/E and I72V amino acid residues relative to .SEQ ID NO:9 when aligned by protocol I or protocol 2.
In a further embodiment, the disclosure provides compositions, comprising one or more of the polypeptides of any embodiment linked to a scaffold. In one embodiment, the scaffold comprises a protein scaffold. hi a further embodiment, the polypeptide is covalently linked to a protein subunit of the protein scaffold to form a fusion protein.
In various further aspects, the disclosure provides nucleic acids encoding polypeptides or fusion proteins of the disclosure; expression vector comprising the nucleic acids of the disclosure operatively linked to a suitable control sequence; host cells comprising nucleic acids, expression vectors, and/or polypeptide or fusion proteins of the disclosure, and pharmaceutical compositions or vaccines, comprising (a) one or more of the polypeptides, composition, nucleic acid, expression vector, and/or the host cell of the disclosure; and (b) a pharmaceutically acceptable carrier.
In another aspect, the disclosure provides methods for treating or limiting development of an influenza infection, comprising administering to a subject in need thereof an amount effective to treat or limit development of the influenza infection of a polypeptide, fusion protein, composition, vaccine, nucleic acid, expression vector, host cell, pharmaceutical composition, and/or vaccine of any preceding claim.
Description of the Figures Figure 1(A-B). Overview of Influenza Neuraminidase design and expression of soluble NA protein. (A) Schematic representation (14.1N1 California 09pdm) of( 1) wild type (NT) and two recombinant NA designs (2 and 3), that vary in amino acid length.
The stalk domain of the WT was replaced by a 6xHis-tag, a IA/SAP domain for protein tetramerization

5 (TD), a thrombin site and a GO linker. (B) Gel filtration chromatograms of His-purified H IN! California 09pdin recorded at 280 nit wavelength.
Figure 2. Characterization of NA subtypes. Different subtype NM differentially 'adapt to open and closed. tetramers: A/Calitbrnia107/2009 Hi Ni, AiNew Caledonia/20/1999 HINI, A/Michigan/45/2015 HINI, AIWSN/1933 H..IN1 and A/Sichuan/26221/2014 Ii5N6 are open. A/Netherlands/219/2003 F17N7, Aniangxi-Dongh046-212013 HI MS and A/Anhui/I/2013 117N9 are mixed open/closed. AAVisconsin/6712005 14.3N2õ
A/Swine/Missouri/2124514/2006 112N3, AiRed knot/Delaware Bay/310/2016 HI0N4 and A/Shorebird/Delaware Bay/30912016 HION5 are closed.
Figure.3. Design process Of /X./California/07/2009 HINI NI neuraminidase, from loose to tight tetramers. Design 94 (SEQ ID NO:79) with ten different mutations resulted in 45% tight tetramer particles. Design .113 (SEQ ID NO:SI ) with 1.1 different mutations resulted in-80-90% tight tetramer particles. Design 155 (SEQ ID NO: 13) with ten different mutations resulted in 90-100% tight tetramer particles.
.15 Figure 4. Design process of AiMichigani45/2015 .H.INI NI
neurarainidase, from loose to tight tetramers based on A./California/07/2009 HINI NI. design.
Design 300 (SEQ
ID NO:14) with ten mutations 'based on design 155 resulted in 75% tight tetramer particles.
Design 174 (SEQ ID NO: .18) with one additional mutation resulted in 100%
tight tetramer particles. Three other distinct solutions for stabilizing mutations were found as well.
Figure 5. Design. process of A/WSNII933 HINI NI ncuraminidase, from loose to tight tetramers based on AiCalifornia107/2009 HINI NI design c155. Design 366 (SEQ ID
NO:14) with 16 different mutations resulted in 80% tight tetramer particles.
Ten of those mutation are the same mutations as in design c155 (SEQ ID NO:13).
Figure 6. Design process of AlVietnain/I203/04 FI5N1 NI ncuraminidase, from loose to tight tetramers based on A/California/0712009 H IN Ni design c155. Design resembles same mutations as design 155. Design. 354 (SEQ ID NO:40) with .14 different mutations resulted in 100% tight tetramer particles. Ten of those mutation are the same mutations as in design c155.
Figure 7. Design process of Alliamixi-Dornthu/346-212013 HIONS NS
neuraminidase, from loose to tight tetramers based on AiCalifo:mia/07/2(X/9 HINI Ni design. Design 285 (SEQ ID NO:36) with two space D mutations resulted in 100%
tight tetramer particles_ Figure 8. Design process of A/Wisconsin/67/2005 115N2 N2 neuraminidase, to de-stabilize the tight tetrameriepartieles based on A/California/07/2009 HIN I Ni design.

Introducing two mutations in design 157 did not result in an open construct.
Adding one additional mutation 165Q resulted in 0% tight tetramer particles with sonic unassembled subunits.
Figure 9. BLASTp alignment of SEQ ID NO:I with other N1 strain sequences that contain deletions or insertions. BLASTp alignment allows for residue positions of reference strains to be matched with corresponding residue positions in strains from the same subtype, even when sequences contain arbitrary numbers of insertions and deletions relative to the reference strain.
Detailed Description All references cited are .herein incorporated by reference in their entirety.
Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al..
1989. Cold Spring Harbor Laboratory Press), Gene Exprenion Technology (Methods in 'Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcerõ
ed.., (1990) Academic Press, Inc.); Pad.Protocols: 4 Guide to Methods and Applications (Innis, et al 1990. Academic Press, San Diego, CA), Cuiture of.Ananal Cells: A. Manual 4.13,asie Technique. 2L'dEd. (R.1. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-12$, ed. E.1 Murray, The Humana Press Inc., Clifton, NJ.), and the Ambion 1998 Catalog (Ambion, Austin, TX).
As used herein, the singular forms "a". "an" and "the" include plural referents unless the context clearly dictates otherwise.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asti; N), aspartie acid (Asp; D), atvinine (Arg; eysteine ((ys;
C), glutamic acid (GIu.; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isokucine (1k; 1), leucine (Len; L), lysine (Lys; K), .methionine (Met; M), phenylalanine (Phe;
F), proline (Pro; P), serine (Sex; 5), threonine (Thr; T), tryptophan (Tip; W), tyrosine (Tyr; Y), and valine (Val; V).
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and. the like are to be construed in. an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Words using the singular or plural number also include the plural and singtilar number, respectively. Additionally, the words "herein,"
"above," and "below" and words of similar import, when used in this application, shall refer to this 'application as a whole and not to any particular portions of the application.
The disclosure provides non-naturally occurring mutant neuraminidase (NA) polypeptides that improves expression and/or modifies the open/closedtetrameric conformational state of the NA polypeptide. As detailed in the examples that follow, the inventors have produced recombinant NA polypeptides in Which head domains comprising stabilizing mutations are connected to tetramerization domains. We initially found that many 'wild-type sequences of beta propeller head domains from certain NA subtypes adopted "open" conformations in which the head domains extended individually off of the stalk-like tetramerization domain, without forming the crystallographicall y observed.
"closed" tetramer.
Constructs comprising the head domains from other NA subtypes formed closed tetramers naturally. Similar constructs from yet other subtypes formed mixtures of open and closed tetramers. We identified specific mutations at multiple locations in NA
sequences that dictate the open or closed .conformational state of NA tetramers, and used these mutations to generate closed, stabilized tetramers from multiple NA subtypes. We also converted a naturally closed NA tetramer to a fully open conformation by substituting residues that we identified as pivotal for tetramer closure, confirming the importance of these residues for determining the confomiatitmal state of NA. Monoclonal antibodies (nAbs) that bind across the interface of two neighboring protomers in the closed --configuration bind better to closed tetramers than open tetramers. The NA polypeptides of the disclosure are improved vaccine antigens in either soluble form or When presented on scaffolds.
In a first aspect, the NA polypeptides are:
(a) at least 75%, 80%, 85%, 90%, 9.1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the Ni NA polypeptide of SEQ. ID NO:1, wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, or all 7 of the following amino acid residues relative to SEQ ID NO:1 when aligned by Protocol 1 or protocol 2:
161T/A, 105A, 165T11, 166P, 196Q, 203Y, 444V ; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the Ni NA polypeptide of SEQ ID NO:1, wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the following amino acid residues relative to SEQ ID NO:I when aligned by protocol 1 or protocol 2:

161 VA/WS% :1001_, 408M, 419V, 9911, 103N, 105.A, 131QINI, 1631/Lõ 165TISMAIL
166P, 1771, 196Q/T, 203Y, 2051, 4421, 444V
Lit tins first aspect the NI NA .ñ fret sequence is based on Ni reference strait A/CalifOmia.107/2009 from H I NI.
MNQKiITiVCNTiCWdLI.LQiGNI rSiwI S HS I QLGNQNQ I ETCNQ S VI TYENTITWVNQ
TYVNT
FitgiCOSVVSVKLAGNS LCWSGWAI YSKDNSVRI G SKGDVFVIRE PFI SC S PLE
CRTFFLTQGALLNDKHSNGT I KDRS P YRTLMSC P I GEVPS PYNSRFE SVAWSASACHDGI NWLT
IGISGPDNGAVAVLKYNGI I TDT IKS WRNN ILRTQE SE CACVNGSC FTVMTD GP SNGQAS YKIF
RIEKGKIVKSVEMNAPNYHYEECSCY PDS SE I TCVCRDNWHGSNRPWVSFNQNLEYQIGY I C SG
FGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWI GRTKS I SSRNGFEMIWDPNGWTGTD
MIPS IRQD IVG INEWSGYSGSFVOR PELTG LDC IIIPCFWVEL IRGRPKENT INATTSGSS IS FCGV
NSDTVGVISWPDGAELPrxIDIc wherein the hold regioil is the head region Odle Ni HA protein As used throughout this application (14 all NA subtypes), 'Protocol 1" and "Protocol 2" both permit alignment of polypeptide against the reference sequence (SEQ ID
NO: 1 in the above embodiment; SEQ ID NOs:1-9 in embodiments described belpw), taking insertions and deletions into account. Thus, the percent identity requirement is based on alignment with the referenee sequenee While discOunting inser:ions or deletions relative to the reference potypeptide.
Protocol I
To run a BLASTp alignment :Online, use the National:Center for BiotechnOlOgy Information (NCOI) server (Or see the article https://Www.nebi,Mm.nib.goviptnelarticies/PVIC14691.11).
a. littpS;f7h1ismehi v/Bla st teinS
2. Set up a BLAST alignment using the following settings:
-1J.Se the option for oAtign two or more sequences!' -Enter the subtype-spoelficreference Wain sequence for the relevant NA
subtype into the "Enter Query Sequence" section -Enter any sequence of the sameNA subtype into the "Enter Subject Sequence" section.
gori thin: blastp (proteinr-protein BLAST) -Expect threshold: 0.1 -Word size: 6 -Max matches in a query range: 0 -Matrix: BLOSUM62 -Gap costs:
-Existence: 11 -Extension; I
-Filter bow complexity regions?: No -Mask:
-For lookup tableonly?: No -Lower case letters?: No 3.. Run the analysis by clicking the "BLAST" button 4.. Click on the "Alignments" tab to show the alignment between the two sequences 5. For each sequence position of intereSt identify the number according to the "Query" sequence. Then identify the corresponding residue position in the "Skier sequence that has been aligned with the position of the "Query" sequence.
Protocol 2 I To run a protein BLASTp alignment on a personal computer or server, install BLAST for command line execution or identify a computer or server that already has it installed.
a. Directions for installation can be found in the user manual:
"BLAST''' Command Line Applications User Manual", National Center for Biotechnology Information (US). Bethesda, MD. 2008.
littps:Siwww.nebi.nlm.inh.govibooksiNBK279690/
2. Generate a file in PASTA format that contains the desired subtype-specific reference strain for the relevant NA subtype. In the command below, this .file will be named "query.faste.
3. Generate a second file in FASTA format that contains an NA sequence of interest from the same subtype, in the command below, this file will be named "sbjet.fasta".
4. Execute the following command using a program such as Terminal, iTerm2, Windows Console, Linux console or other similar terminal emulators. This will.

generate results in a file named "results,txt".

blattp -query query.fasta -subject sbjet.fasta -matrix BLosume -evalue -word_size 6 -gapopen 11 -gapextend I -out results.txt 5. Open results.txt and view the section showing alignment of the two sequences.
For each sequence position of interest, identify the number according to the "Query" sequence. Then identify the corresponding residue position in the "Shia"
sequence that has been aligned with the position of the. "Query" sequence.
in another embodiment of any aspect, embodiment or combination of embodiments described herein (for all NA subtypes), the percent identity is based on an alignment of the polypeptide to the reference sequence using any protocol, and insertions and deletions relative to the reference poly-peptide are not considered in determining percent identity. In a further embodiment or combination of embodiments described herein, the percent identity is based on an alignment of the polypeptide to the reference sequence using any protocol, and insertions and deletions relative to the reference polypeptide are considered in determining percent identity.
Throughout the application (for all NA subtypes), mutations that primarily improve expressio.n a the engineered NA polypeptides arc denoted in bold font.
Throughout the application (for all NA subtypes), mutations that primarily destabilize the engineered NA =enters are denoted in italicized font.
In one embodiment of this first aspect, the polypeptides are (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the NI NA polypeptide of SEQ ID NO:11 , and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, or all 7 of the following amino acid residues relative to SEQ ID NO:I when aligned by protocol 1 or protocol 2:
(i) 16117A, 105A, I57K, 165171, I66P, 196Q, 203Y, 444V; or (ii) 105A, 165T/I, 166P, 196Q, 203Y, 444V.
In another embodiment of the firstaspect, the polypeptides are (b) at least 75%, 80%,.85%, 90%, 91%õ 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the NI NA polypeptide of SEQ ID , and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the following amino acid residues relative to SEQ ID NO:I when aligned by protocol I or protocol. 2:

i(i) 16ITIAN/SIT, 100L, 408M., 4 I9Võ 99P, 103N,.1.05A,131Q/M, 1631/L, 1.-.65T/SMAII, 66Põ.1771, .196Qaõ.203Y,. 2051, 4421, 444V; or (Al) 99P; 103N, .105A,, 13 IQ/M, 1631/L, 165T/SiViAl1, 166P, 1771, 196QTL
203Y.:.205.1õ.4421, 444V, in a further &Ohba-nerd- of this first aspect, the polypeptides include one or more of the. following sets of amino' ncid rosidues.rolative to. SEQ. ID NO when atigued by protocol 1 or protocol (1) 161V/172A;
100L/408M;

103N/105A;
1141/166P;
101C/164C;
101C/164C/174V;
101C/122V/164C/174V/444V;
I 22V/444-V;
131Q/442I;
101A/163L/444A;
131M/4421;
101A/131M/163L/4421/444A;

131M/163L/4421/444A;
100L/131M/163L/4421/444A;
99P/1001.1161V/165.S1172A/1.77V196T/2051/408M/419V;
991/ 1=001.11.61W1.6.5S1172A/1.77V1.96T/408M/419V;
99P/100L/131Q/161W165S/172A/1771/196T/20511408M/41.9V;
99P/100L/131Q/161V/165S/172A/1771/196T/408W419V;
99P/100L/101A/131M/161V/163L/165S/172A/1771/196172051/408M1419V/44211444A;
99P/I.001-1101A/1.31:M/161V/1631/165S/172A/1771/196T/408M/419V/4421/444A;
99P11.61V11.65SII 72Al I 7'7111.96T/2051;
99P/161V/165S/172A/177111961%
99P/131Q/161V/165S/172A/1771/196T/2051;

99P/131Q/1 61 65S/172AI 1 771/196T;
99P/10 1A/131M/161 V/1631J1 65511-721101 771/1 96T/2051/4421/444A;
99R1101 Ai 113 1 MI 161 V/16311165571.12A/1771,1 96T14421/444A;
99P/196T;
99P/196T/2051;
99P/ 1771/190T; and/or 99P/1 771/.I 96V2051; or 103N/105A;
1141/166P;
101C/164C;
101C/164C/174V;
101C/122V/164C/174V/444V;
122V/444V;
131Q/442I;
101A/11 63L/444A;
131M/4421;
101A/131M/163L/4421/444A;
100L/101A/131M/163L/4421/444A;
131M/163L/4421/444A;
100L1 1 3 !WI 63Li442,11444A;
9911( WW1 01 W165S/ 1 72A/1 771496T/20511408M141 9V;
-99P/1001_1111Q/161Vil 65S/172A/1771/196T/2051/408M/419V;
99P/100L/ I 31Q/16IV/165S/ I 72A/1771/196T/4I9V;
99P/1001,1 tA/1311\41161V/1 631.1.105S,'172A/1771/196T/2051/408M/419V/4421/444A;
99P/1Q01,11014/131M/16.1V/1631,1165S/17qA/1771/196T/408M/419V14421/444A;
99P/ 1.6 1 V/ 165$/ 1 72A/ I:77 V 1 96T/2051;
991)1161 V/165 S/1 72N 17711196-1;
99P/131Q1161V1 65S1172A117711196172051;
99P/131.Q/ 1 6.1 V/1.6551.172A/ 1 77.11.1 96T;
99P/101A/131101101V/163U165S/172A/1771A96T/2051/4421/444A;
99P/1 01A113 1.W1 V/1631.1165S1 72A11.771,4 96T/44211444A;
99P/196T;
99P/196172051;

99P/1771/196T;:andlor 99P/1771/196T/2051_ various embodiments, of this :first aspect, the :polypeptide.s: inc1udes:3, 4, 5, 6., 7,S, 9 or more of the listed amino acid msidues relatiVe to SEQ IDNO:1 When aligned by protocol 1 or protocol 2.
In a further eniboditnent, the pelypeptides further comprises I2,3, 4, 5., or all 6: of the following residues relative to SEQ ID NO:.! When aligned by protocol I or protocol 2:
1055, 106V, 1631, 166V, 210G,: 4425, IQ In a still furtha embodiment of the first aspect, the polypeptides include one or more of the fellowing sets of amino acid residues relative to SEQ 1.1) NO:1 when aligned by protocol) or protocol 2:
99P11.001,1161V/1655/172 A/1771/196Tp,051/408M/41 I 9V;
99P/10011,1161V/165S1172A/1771/196T1408W419V;
99P/100L/131O/16I VII 65S1172A/1771/196T12051/408M/419V;
99P/100L/131Q/161V/165S/172A/1771/196T/408M/419V;
99P/100L/101A/131M/161V/16311165S/172A/1771/1961/2051/408M/419V/4421/444A;
99P/100L/101A/131M/161V/163L/165S/172A/1771/196T/408M/419V/4421/444A;
99P/161V/165S/172A/1771/196T/2051;
99P/161V/165S/172A/177U196T;
99P1131Q(.161V/165S/172Al1 771/196172051;
99P/131Q/161V/1658/172A11771/196T/;
-99P/1.01 AI13 IMJ16111/1631,1165$1172A/1771/-196T/2051/4421/444A; and/or 99P/101A/131W161V/16311165S1172A/1771/196T/4421/444A.
In a stilt itiatber embodiment, the polypeptides comprises an aniinp acid sequence at Nast 75%, 80%, 85%, 90%, 92%, 93i),. :94%, 95%, 96%, 97%, 98%, 099% identical.
to the amino acid selected from the gilyup consisting of NI mutants listed in Mt* I (SEQ ID
NO;! 0-32 and 39-95), when aligned by protocol I or protocol 2, wherein the polypcptide includes all of the reSidnes listed in Table 1 for an individual Ni mutant listed in Table I., Inasecond aspect, the NA polypeptides are:
(a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9.5%, 96%, 97%õ 98%, or 99% identical to the N2 NA polypeptide of $EQ. ID NO:2, and wherein the non-nnturtilly occurring polypeptidc includes 1, 2, 3,4, 5, 6, 7, or all 8 of the followirm arninp acid residues relative to SEQ ifl :NO2 when aligned by protocol I or protoeol 101C,, 131M, 162P, 165S11. 166V, /95Q, 202Y, 443S: Or (b) t least 75%õ /0.4, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 90%, 97%, 98%, or 99% identical to the N2 NA polypeptidc of SEQ ID NO:2, and wherein the non-naturally occurring po1ypeptide includes 2, 3, 4õ 5, 6, 7, 8, 9, 10, 11, or all 12 of the following: amino acid residues relative to 'SE() 10 NO:2 when aligned by protocol 1 or proopeci12;
101(.71A, 103-N1, 105A, 106V, .1 ;11!,1, 164", 1631,165S/11M, 166v, 195Q, 292Y, 443Sõ
thIs second aspect, the N2 NA refektmed sequence is based on N2 reference strain 19 AlWiscOnsint67,2905 from -113N2.
PNQX I IT I G$VSLT STICFMI AIL I TTYT:LliFKQYETtisPiniPAVNILCE.T.-"r rvy 'NTT T FTETC PFLAEYRNWSKPQCNITGPAPPSICDNSIRLSAGGDItiVTREPYVSCDPDKCYQFALGQ
GTTLNNVHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSS SSCHDGKAWLHVCVTGDDKNATAS
FIYNGRLVD S IVS WSKE I LRTQE SECVC ING T CTVVMTDG SAS GKAD TKI L F IEEGK I VHT
S TL S G SA
QHVEECSCYPRYLGVRCVCRDNWKGSNRP IVD INIKDYS I VS SYVC SGLVGDTPRKND SSSS SHCLD P

NNEEGGHGVKGWAFD D GNDVWMGRT I S EKLRS GYET FKVI E GWSNPN SKLQ I NRQV IVDRGNRS
GY S G
I FSVEGKSC INRC FYVEL IRGRKEETEVLWT SNS IVVFCGTSGTYGTGSWPD GAD INLMP I (SEQ
ID NO:2), wherein the highlighted residues are the head region in one this second aspect, the polypeptides are:
(a) at feast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9$%, or 99% identical to the N2 NA pelypeptide of SEQ 10 NO:2, and wherein the non-naturally occurring polypeptidc includes 1,2, 3,4, 5, 0, 7, or all of the following amino acid residues relative to SEQ ID. NQ:2 *hell aligned by protocol 1 or protocol 2:
101c 131M, 162P, 105V1I, 166V, 195Q, 202Y, 443S_ In another 'embodiment, ttie polypeptides are:
(b) at.IcAst 75%, 80%, 85%, 90%, 9'1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypcptide of SEQ ID NO:2, and wherein the non-naturay ocentring:polypeptide includes 2, 3, 4, 5, 6, :Z 8, 9, 10,11, or all 12 of the following amine acid residue's relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2:
101G/A, 103N, 105A, 106V, 131M, I62P, 1631, 165S/17A/1, 166V, 19$Q, 202Y, 443S%

In another embodiment of this second aspect, the polypeptides include one or more of the following sets of amino acid residues relative to SEQ NO:2 when aligned by protocol 1 or protocol 2:
103N/105A;
101C/164C;
10ICI164C1173V;
101A113IM;
1001.,/101A/I31M; and/or 100L/131M/163L.
In various embodiments of this second aspect, the polypeptides include 3, 4, 5, .6, 7, 8, 9, or more of the listed amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2. In another embodiment, the polypeptides of this second. aspect comprises the amino acid sequence at least. 75'.%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid selected from the group consisting of N2 mutants listed in Table I (SEQ ID NO:33-34), when aligned by protocol 1 or protocol 2, wherein the polypeptide includes all of the residues listed in Table 1 for an individual N2 mutant listed in Table I. In a still further embodiment of this second aspect, the polypeptides are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N2 NA .polypeptide of SEQ ID NO:2, and wherein the non-naturally occurring polypeptide includes a 165Q1E amino acid residues relative to SEQ ID
NO:2 when aligned by protocol I or protocol 2, or includes 2 or 3 of 165Q1E, I76V, 195S
amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2.
In a third aspect, the NA polypeptides are:
(a) at least 75%, 80%, 85%, 90%, 91%, 92%., 93%, 94%, 95%, 96%, 97%, 98%., or 99% identical to the N3 NA polypeptide of SEQ. ID NO:3, and Wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all II of the following amino acid residues itiadvs to SEQ 11) NO:3 when aligned by protocol I Or protocol 2:
103N, 105S, 1310M, 157T, 165S/1, 166P, 196Q, 203Y, 2051, 4:13S, 445V; or (b) at least 75%, 80%, 85%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%. 98%, or 99% identical to the N3 NA polypeptide of SEQ ID NO:3õ and wherein the non4iatural1y occurring .polypeptide includes 2, 3,4, 5, 6, 7, 8, 9, 1.0, 11, 12, or all 13 of the following amino acid residues relative to SE() ID NO:1 when aligned by protocol I or protocol 2:

103N, 105S, 106V, I IIQ/M, 157T, 1631-, 165S/1rV/A, 166PIV, 196Q, 203Y, 2051õ.4435, 445V.
nus durd.aspeet, thoN3 NA .rclerenee sequence is based on N3 referencostram A/Swine/Missouri/212451412006 from fl2N3.
MNPticilK ITI=ITTLSTIP.,LLIGVCTILVFNIn'`IFIEKIGDHQIVTYP I ITT PAVPNCSDT I I
TYNNT
VINNI TTT II TEEERPFKSPLPLCPFRGFFPFHKDNAIRLGENKDVIVTRE PYVSCDNDNCWSFALAQ
GALLGTKHSNGT I KDRT PYRSL IRFP IGTAPVLGNYKE I C IAWS S S
SCFDGKEWMHVCMTGNDNDASA
9Ti YGGRMTD S IKSWRICID ILRTQE S ECQC InGTCWAVTD GPAANSADYRVYWIREGK I I
KYENVPKT

TMSCD SPS
NVNGGP GVKG FGFKAGDDVWLGRTVS T S GR S G FE I IKVTEGII INS PNHVI7(S I
TQTLVSNNDWSGYSGS
F IVKAKDC FQPC FYVELIRGRPNKND DVSWTSN S I VI" FCLDNE PeaCkNWPD GSNI QFMPIC
n,t' ad region indicated by highlighting) (SE, ID ,A:3) In one embodiment of this: thirtaspeet, the.pplypeptides (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or 99% identical to the N3 NA OOlypeptide of SEQ ID N0:3, and N.vhereiri theiton,,natutaily.
oceurringipelypeptide includes I,. 6, 7, 8,.9.. i 9; ot all 11 of the follOWiria amino 'acid residues. relative to SEQ. ID NO73.when aligned by protocol 1 or protocol.2::-103N, 1.05$, 131(),IM, I57T, 165511, 1661), 196Q, 203Y. 2051, 443$, 445V.
In another embodiment, the.poiypeptide$:.are (b) at least.75%õ .85%,.90%õ 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N3 NA of SEQID NO.:3, and wberetu the rioh-hatttrally .occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, mall 13 of the. following amino acid rekidues..relatiVere.SEQ ID NO.;3 when aligned by protocol 1 or protocol 2:
103N, 105$, 106V, I3IQM I57T 13L 16.5S/TIVIA, 166P,N,196Q, 203Y, 2051, 443S, 445V.
hi another ernbodirrient Of this third .aspect, the pOlypeptide includes erit.er rriOre Of the following sets of amino acid residues relative to SEQ.ID NO:3 when aligned by protocol 1 or protocol 2:
101C/164C;
101C/164C/174V;
101C/164C/174V/445V;
101A/445V;

101 A/13 1M/445Aõ
13:I M/445A;
11411166P;
101A/163L/445V;
1 01A1131M/1.6311446A runifor 13.1M11.6311445A.
. various further embodiments of this third aspect,: the polypeptides includes 3õ 4, $,

6, 7,8 9: or more of the listed amino acid residues relative to :SEQ.ID NO;3 when aligned =by.
protocol 1 or protocol 2. In another embodiment, the polypeptide. is at least 75%, 80%; 85%, 9 Pfii,.92%), 93 4, 94%,:95%, 96%, 97%, 98%, or 99%. jdcrgical. to the N3 NA
polypeptide of SEQ. II) NO:3., and wherein the non-ma mrally occurring polypeptidernelndes One or both a the following amino acid residues relative to SEQ ID NO:3: when aligned by protocol 1 or protocol 2: 196S, 165Q/E:.
lwarourth aspect, the NApolypeptides are:
(a) at least 75%, 80%, 8.5%,90%, 9.1%,. 92%, 93%, 94%õ:
95%, 96%, 97%, 98%, or 99% identical to the N4 NA polypeptide of SE9 NOA, and Wherein the non-tiaturally Occurring PolYpetilide rncludcs 1, 2,3, 4, .5õ. 6, 7õ3õ 9, 1011õ 12, 13, 14, 15, or all 16 of the followinramino acid residues relative to:SEQ ID:NO:4 When aligned by protbeol 1 or t.isnotricol 1.60V/SITIA, 409M, 102N, 1Q4S(Aõ .105V, 112D, 130Q/M, 162L, 164S/T/All, 165P, 1761, 195Qõ 202Y, 2041, 4431, 44.5V; or i(b) at least..75%,.80%, 85%,.90%,õ91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% *mien]. to the N4 NApOlypeptide of SEO 1D NQ.4,. and. wherein the non-naturally et:wring polypeptide includes 2,3, 4,5, 57, 8, 9, 10, 11, 12,13, 14, 15, or all 10 of the following atnino.:.aeld residues relative to SEQ NO.:4-when aligned by protocol 1 or protocol 2;
1.60V /SIVA, 409M, 102N, 104S/A., .105Võ I I 2D, 130(W, 1621,, 164S/TIA/1, 1651W, 1761, A 95Q,..2,eY:, 2041, 4431, .44.5.V
.30 in this fourth aspect,. the N4 :NA. reference sequence is based on. N4 reference strain inthstoriorDeia.ware.Day/14112016 from H1ON4.
muitstQ.K I -..r.:(3$77.3 T. I MTTVGLIVO 17=5!LCS

NYTT I AE P SAPDVVHYSSGRDLCPIRGWAPLSKDNGIRIGSRGEITEVIREPFISCS I SECRTFELTQG

ALLNDKHSNGTVKDRSPERTLMSCPIGVAPSPSNSRFESVAWSATACSDGPGWLTLGITGPDSTAVAV
LKYNGIITDTLKSWKGNIMRTQESECVCQDEFCYTLVTDGPSDAQAFYKILKIRKGKIVSMKDVDATG
FEFEECSCYPSGTEIECVCRDNWRGSNRPWIRENSDLDYQIGYVCSGIFGDNPRPVDGTGSCNGPVNN
GKGRYGVKGFSFRYGDGVWIGRTKSLESRSGFEMVWDANGWVSTDEDSNGVQDIIDNDNWSGYSGSFS
IRGETTGKNCTVPCFWVEMIRGQPKEKTIWTSGS:SIAFCGVNSDTTGWSWPDGALLPFDIDK
(H4c] 8e,,janc;e hilighted)- (3E0 ID NO:4) in one embodiment of this further aspect, the polypeptides are (a) at least 75%, 80%, -85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N4 NA :polypeptide of SEQ ID NO:4, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8+9, 10,11, 12, 13+14, 15, or all 16 of the following amino arid residues telatiNe: to SEQ. 113 NO:4 when aligned by protocol 1 or protocol 2:
160W/S/1/A, 409M, 102N, 104S/A, .105V 112D, 130Q(M, IL. 104.srum, 165}., 1761, 195Q, 202Y, 2041, 4431, 445V; or OD 102N, 1048/A, 105V, 1121), 130Q/114, 162L, 164S/1-7M, 165P, 1761, 195Q, 202Y, 2041, 4431, 445V.
In another embodiment, the po1ypcpti40$:ai-e 0) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%., 94%, 95%, 90'4, 97%, 9$%, or 999/0 identical to the N4 NA polypeptide of SEQ. NO:4, and wherein the non-naturally occurring polypeptide includes 2,.3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14; 15, or all 10 of the:
following amino acid residues relative to :SEQ 113 NO:4 when 000 by protocol t Or protocol 2:
(i) 160VIS/T/Aõ 409M, 102N, 1048/A, 105V, 112D, 130Q/M, 162L, 16,5Ply, 1161, 195Q, 202Y, 2041, 4431, 445V; or (4) 102N, 104S/A, 105V, 112D, 110Q/K 1624 164SITIM, 165PAT., 1761, 195Q, 202Y, 2041,4431, 445V.
In another embodiment of this fourth aspect the ipolypeptides include one or more of the following sets df amino acid 'residues rclative to SEQ ID NO:4 when aligned by protoodi 1 or protocol 2:
(i) 160VISIVA;
160V/171A;

102N/104A;
100C11 63C;
100C,11630173V;
100C/163C/173V/445V;
130Q/4431;
100A/162L/445A;
130M/4431;
100A/130M/1621144311445A;
130N1/1621-444311445A;
IQ 1761/2041;
1 0Q(7/121 V/163C/ 73V/445V; and/or 21V/445V-, or (ii) 102N/104A;
100C/163C;
100C/163C/173V;
100C/163C/173V/445V;
130Q/4431;
100A/162L/445V;
130M/4431;
I (*iv 139M1162L/4431/445A;
130M/ 621.14431/445A;
1761/2041;;
100C112 1 V/163C/173V/445Y; andlor 121V/445V.
In Amrimts further ernbodimeot Of this fourth avect, the polypeptides include 3, :4, 5, 6, 7, 8,9 or more of the 4ted amino 4cid residues relative to :$EQ ID 'NQ..4 when ifigaed by protocol 1 or protocol 2. in 4 furthet eMbodiiiient, the polypeptide6 are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or 99% identical to the N4 NA
volypdptide of SEC) ID NO:4,:and Wherein the non-haturally declining polypeptide includes 1, or both of the following amino acid residues relative to SEQ NO:4 when aligned by:
protOccit I or protocol 2: 164Q/E, 1955, In a fifth:aspeot, the NA pOlypeptides are:

..at least 75%, 80%, .85%, 90%, 91%, 92%, 93%,. 94%, 95%, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ ID NO.;5, and Wherein the linn-uaturally.
OCCUiliiAt linlypeptide includes 1,2, 3,4. 5, 6, 7, 8, 9,10,11, 12, ur all 13 Of :the.folloWilig.
-amino neid residues relative to SEQ ID NO:5 when 'aligned by protocol I or protocol .2:
971,, 410M, 961:); 98Aõ 100N, 102S/A, 1 I OD, 128Q/1g, 1601,162V/AiT, 1.63V/P-1931)/T;
4451; or (b) at /east 75%,:f30%, :85%, 90%,.91%, 92%, 93%, '90'495%, 96%, 97%, or 99% identical to the N5 NA.polypeptide Of SEQ ID NO:5., and wherein the non-naturally.
occurring Polypeptide includes .2, 3, 4.5,. 6, 7, 3, 9., 10, 11, 12,1.3, 14, 1.5, or all 1-6.ofth0 foliowirtg Amino acid residues relative SEQ 113. 1'40:5 Wherizliftied by protocol 1 or protocol 2:
97L, 410M, %/1, 98A, 190N, 102S/A, 103V, 110D, 12kQfkl, 154T, 1601, .162V/Ailtrõ
63WP , 1741, 193Q/T, 4451 In this fifth aspect, the N5 NA reference sequence is based on N5 reference Strain AlgulliDelaware Bay/2.18/2016from .1.110N5, Plgi,;,=11 IT T.
GsysT,4ktyyrNILIMIA,S,IVIG.I.I"rs=PTIKET..M.4.:STCNTTEVY.NE:17VRLFT TTPINNT
V.Y1ERESEQFPEPLNISTTEPLCNVSGFAIVEKDNGIRIGSRGIIVFV1REPFVACGPTECRTPFLTQSAL
LINTOKRSNNTVKDRSPYRALMSVPLGSSPNAYQAKFESVAWSATACHDGERVILAVGISGADDaNYAVIR
YGGMPTI3VVRZWRKQILRTQESSCVCMKGNCYWVVITDGPANSQASYKIFKSHKGMVTNEREVSFQGGli IEECSCYPNLGKVECVCRENisTNGMNRPVLTFDEDLNYEVGYLCAGIPTDTPRVQDNSFIGSCTNAVGG
SGTNNYGVKGPGPRQGNSVWAGRTVSISSRSGFEILLVEDGWVKTSKIsIVVIUWEVLNNENWSGYSGAF
TIPITMTSKQCLVPCFWLEMIRGXPEERTSITITSSSSTVFCGVSSEVPGWSWDOGAILPFDIDKM
(Head is 4ig4ig4.) (SEQ. ID NC,:t.:0 in one embodiment of this fifth aspect, the polypeptides :are (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N5 NA .polypeptide of SEQ ID lsit?:5, and wherein the non-naturally meurring..polypeptide includes 1, s2, .3, 4, 5õ.6, 7, 9, 1%11, 12, oral] 13 ofille.follOwing aminaaelci residues relative to .$134) NO:,5 when aligned by protocol 1 or protocol..27 410M, 96F% 98A, 100N,. .102S/A, 110D, 128Q/M, 1601, 162V/A/1, 163V/P, 1.93Q/Tõ 4451; or 961),..98A,. 100K 1025/A, 110D, I 281)1M, 1601. 1 62WAIT, 1.63V/P, I 9 3Q/T, 4451.
In another embodiment of this fifthaspeet,:the polypeptides are 2:1 (b) At Icat 75%, 80%, .85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ11) NO.;5, and Wherein the tinn,naturally.
At **peptide iticludeg 2., 3, 4, .5, 6, 7, 3,, 9, 10, 1.1, 12, 13, 14, IS, or ail 1.6 Of the followirigaminoacid.regidues relative to .SEQ. 11) NO:5 whenaliinted by protocol I. or protocol 2:
.(4.) 97L, 41.0M:, 96P, 93.A, 10.0Nõ 102S/A, 103V, 1101), 128Q/M, 154T, 1601, I 6.2V/AI1/T, 1.03\i/P 1741,193qT,.4451; or 96P, 98A.,.10ai, 102SIA,.103V, 110D, 128Q/M, 1541, 1601, 162VIAIFT, 163VT, 1741,193QT 445L
in ether einhodinients of this fifth aSpect, the .polypepticies irtelwle one or more of the following. set$ olannno acid residucs..telative :to SEQ ID N.Q.:5 when aligned. by protocol 1 or protocol 2:
97L/410M;
100N/102A;
98C/161C;
128Q/445I;
128M/445I;
98A/128M/4451/447A;
97L/98A/128M/4451/447A;
1.2WW4451/447A;
971,11.28M144511447A.;
96P/193T;
I111/163P;
96P/1.93T/202i;
96P/1 7411193T: or 96P,!'1.741/1.93172021; or 100N/102A;
98C/161C;
128Q/445I;
128M/445I;
98A/128M/4451/447A;
97L/98A/128M/4451/447A;

1.28M/4451/447A;
971j128Mi4451/447A;

1111/163P;
96P/193172021;
96p/1741/19,3j; and/or 90Pii 74093T/2021.
In further embodiments of this fifth aspot, the polypeptides includes 3,4, 5, 6,7. :8, 9 or more of the listed amino acid residues relative to SEQ tO NO:5 when aligned by protocol 19 1 or protocol 2. In a fUrther embodiment, the polypeptides are at. leaSt 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the V: NA
polypeplideof SEQ ID NO:5, and wherein the non-naturally occurring polypeptide includes:1 , or both of the -faii0Wing Wning acid residues relative to SEQ Lfl NO µ,yhert aligned by protocol I or protocol 2: 162 QiE:, 209S, In a sixth aspect, the NA polypepOdes 41;17q:
least 75%, 80%, 85%, 90%, 9:1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or 99% identical to the N6 NA poly0eptide of SEQ ID NO:6; and wherein the non-naturally oecurring,polypentide includes 1, 2, 3,4, 5, 6, 7, k 9,10,ot all 11 of the following amino add residues relative to SEQ ID NO:6 when aligned by protocol .1 or protocol 2:
99P, 193N, 113D, 131.Q, 1611, 162P, 1631, 165STIV/A, I96Q, 203.7.1, 445S; ot .(b) at least 75%,, 80%, 85%, 9.0%,9,1%, 92%, 93%, 94%, 95%, 96%, 97%, qs%, or 9" identical to the. NO NA poly-peptide of SEO ID NO:6, end wherein the non-nntutally occurring polypeptide includes 2.3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following ntnincs acid residesmlative to SEC) ED NO.:6 when aligned by protocol: 1 or protocol 2:
99P, 103N, LOS, 106V. 130, :1.1Q, 157T, 161I, 162P, .1631/1., 165S/TN/Ail, .166V1P, 196Q, 2PY, in this sixth aspect the N6 NA. ram= sequence is tvs03 on N6 reference strain Alchickett/SithuaniNCIPL112014 from H5N6.
PN TC S AT GMT LS WSAL IG AULGLN IG rAirlYEMS D 2, TN IN I
PIIMN E TS PT TT II NNtiPQNNF

GT TLRGKHANGT I HDRS PFRALVS WIEMGQAPSPYNTRVEC I GWS S T SCHDG SRMS IC IS
GPNNNASA
VVWYGGRPVTE I P SWAGN I LRTQE S E CVCHGG I CPVVMTD GPANNRAETKI I Y FKEGK I KKI
EELKGD

AQHIEECSCYGASEMIKCICRDNWKGANRPVITIDPEMMTHTSKYLCSKILTDTSRPNDPTNGKCEAP
ITGGSPDPGVKGFAFLDGENSWLGRTISKDSRSGYEMLKVPNAETDTQSGAISHQIIVNNQNWSGYSG
AFIDYWANKECFNPCFYVELIRGRPKESSVLWTSNSIVALCGSKERLGSWSWHDGAEIIYFK (Head aleence hi'ghlighted) (SEQ TD NO:f;) in one embodiment.of this sixth .aspect, the polypeptides are:
(a) .at. least 75%, 80%õ .85%, 90%, 91%, 92%, 93%,..94%, 950, 96%,..97%, 98%, or 99% identical to the N6 NA polypeptide of SEQ N.0:6; and wherein the non4taturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9õ10, mall Ii of the following amino 0 acid residues relative to SEQ. 1.D.NO.:.6when:aligned- by iprotocol 1 or protocol.2:
99P, 103Nõ 113D, 131Q,-1611, 162P, 1631, 165SITNIA, 196Q, 203Y, 445S.
In another embodiment (alibis sixth aspect, the polypepti des are:
(b) at least 75%, 80%,-85%,:90%, 91%, 92%, 93%, 94%õ95%, 96%, 97%,. 98%, or 99% identical to the N6 NA polypeptide of SEQ ID NO 6 rid whemin the...non-naturally 115 occurring polypeptide includes 2, 3, 4, 5, .6, 7, )Jõ 9, 10, 11, .12,11, 14, or nil 15 Of the following amino acid residues relative to SEQ 113.N0;6 when aliened by protocol I or protocol 99e, 103N, 105S, 106V,11.3D, 131Q, 1571, 1611,162P, .163.111, 165$1TN/A11õ
166WP, 1960, 203Y, 445&
20. In various further embodiments of this sixth aspect, the polyveptide include one or more of following sets of amino acid residues. relative to SEQ ID NO:6 When alistied by protocol 1 or protocol 2:
101C/164C;
101C/164C/174V;
25 101C/164C/174V/447V;
122V/447V;
1:01A/163L;
99P/1961; .andlor 99P(196P2051.
30 In further embodiments, the polypeptides Include 3õ 4, 5õ 6, 7, $õ.9 or more of the listed amino acid residues rclatiµT to SEQ ID NO:6 when aligned by protocol 1 or protocol 2..
In another embodiment, the polypeptides arc at leaSt 75%, 80%, 85%, 90%, 9.1%, 92%, 93%, 94%õ 95%, 96%, 97%,:98%, Or 99% identical to the N6 NA polypeptide of SEQ ID
N06.,.
wherein the non-naturally Ottkirring tuAypOptide inichidOS. a. I65E
arnirfOacid. mutation teiati'v to SEQ ID .NO6 when aligned hy protocol 1., and optionally also includes a 177V
amino: acid trintatitin relative to SEQ ID Na6 when aligned by 'protocol 1 or -priatticOl 2, In a seventh aspect, the NA: polypepiides: are:
(a) at least 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95% 96%, 97%, 98%
or 99% identical to the N7 NA polypeptide Of SEQID NO:7, and wherein the lion-natural-4F
occurring **peptide oiclude 2 3, 4, 5, 6, 7, 8, 9,1.0, orall 11 of the following amino acid residues relative to SEQ ID NO:7 when aligned by protocol I or protocol 2:
98P, 102N, 1045, 1121), 130Q, 156T, 1:621, 164V/A11, 165V, 202Y, 448V; or (h) at 104 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the Ni NA polypepticle of SEQ ID NO:7, and wherein the non-naturally omitting poly-peptide includes 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, or all 13 of the following -amino acid residues relativetb.SEQ ID NO:7 when aligned by protocol or protocol 2:
98P, 102N, 104$, 112D. 130Q, 156T, 1621, 164V/All, 165V, 1761, 202Y, 4461, 448V, 1 5 In this seventh aspect, the N7 NA reference Sequence is based on N7 reference strain AiNetheliands/219/2003 from HINT
MNPNQKLEALSGVATALSVLNLLICTSV=MVSLHLKEYGPKQEENLTCTTINQNNTTVVENTYVNN
TTITTRGTDLKTPSYLLLNKSLCNVEGWVVIAKDNAVRFGESEQIIVTREPYVSCDPTGCKMYALHQG
TTIRNKHSNGTIHDRTAFRGLISTPLGTPPTVSNSDFMCVGWSSTTCHDGIARMTICIQGNNDNATAT
VYYNRRLTTTIKTWARNILRTQESECVCHNGTCAVVMTDGSASSQAYTKVMYFHKGLVVKEEELRGSA
RHIEECSCYGHNQKVTCVCRDNWQGANRPIIEIDMSTLEHTSRYVCTGILTDTSRPGDKSSGDCSNPI
TGSPGVPGVKGFGFLNGDNTWLGRTISPRSRSGFEMLKIPNAGTDPNSRIAERQEIVDNNNWSGYSGS
FIDYWNDNSECYNPCFYVELIRGRPEEAKYVWWASNSLIALCGSPFPVGSGSFPDGAQIQYFS
(Head sequence highlighted) (SEQ ID NO:7) :in one embodiment of this seventh aspect, the polypeptides are (a) at least 75%, 80%, .85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N7 NA **peptide of SEQ ID NO:7, and wherein the non-naturally occurring .polypcptide includes 1,2, 3,4, 5,6. 7, f$; 9,10, or all 11 of the following gun*
acid resWttes relative to SEC,/ ID NO:7 when aliped by protocol 1 or protocol.
981), IO2N, 104$, 1121), 130Q, 1561, 1621, 164V/A/I, 165V, 202Y, 448V.
In another embodiment, the **peptides are (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N7 NA polypeptidc of SEQ ID NO:7, and wherein the non-naturally Orem:ring polypeptidc includes 2, 3,4, 5, 6, 7, 8, 9,10, 11, 12, or all 13 of the following amino acid residues relative: to :SEQ ID NO:7 when aligned by protocol I or protocol 2:
98P, 102N, 104S, II21), I 30Q, 156T, 1621, 16W/At!, 165V, :1761, 202Y, 4461, 448Võ
In various further einbodinlents of this seventh aspect, The polypeptide includes one or more of the fbIlowing sets of amino=acid residues relative to SEQ ID NO:7 when aligned by protocol 1 or protocol 2:
100C/163C;
100C/163C/173V;
100C/163C/173V/448V;
99L/4461/448A;
98P/1951;
130Q/4461;
4461/44M;
98P/195T/2041;
98P/1761/195T; And/or 981)/I761/195172041õ
in further embodiments, the pOlypeptides include 3,4,5, 6, 7, 8, 9, Or more of the listed amino acid residues relative to.SEQ 10 NO:7 when aligned by protocol 1 or protocol 2.
in another embodiment, the potypeptides are at least 75%, KV* 85%, 90%, 91%, 92%, 9÷.=;, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N7 NA. polypeptide of SEQ ID
NO:7, and wherein the On-naturally occurring polypeptide includes-one or bOth of the following minim *id mutation relative to. SEQ JD NW when Aligned by protocol I or protocol 2:
164Q1E, 195&
in an ..eighth aspect,: the NA polypeptides are:
(a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide Of SEQ.: ID NO:8, and wherein the TICit-linturzilly occurring polypeptide includes , 2, 3, 4, 5, 6, 7, .8, 9, 10, Oran 11 of the following amino acid residues relative to SEQ IDNCY8 when aligned by protocol I or protocol 4081, 10.11NI,103ASS, 1110, 129Q, 161P11, 163S/TN/AIL 164V, I 94Q, 201V, 4421;
:or qy) at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA .polypeptide of SW: ID NO:8, and wherein the non-naturally occurring polypeptide includes 2,3, 4, .5,6. 7, 8, 9, 10, I 1 , 12, 13, 14, or all 15 of the 2:6 follOwittg amino acid residues relative to SEQ NO:8 when aligned by protocol 1 or protocd 981, 408M, JOIN, 103A/S, 104V, 1110, 129Q/M, 161117L, 1635ITIVIA11/ST, 164V1P, 1751,, 194Q, 20 Y, 2031, 4421 In this eighth aspect, the N8 NA reference sequence is based on N8 reference strain:
Adi PB13b/2013 from Hi QNS.
1.1ti ?Nr:e=KI T IGSV$Lec Lvi TAN ELIA V.$ TVT VILvL P GNG N NE s CNETV RE
YNETVRVEIWTQIIHNT
WITEYTFRMKrDHEMNNTEALCDAKGFAPFSKDNGIRIGSRGHVFVIREPFVSCSPTECRTFFLTQGS
LLNDKHSNGTVKDRSPYRTLMSVEIGQSPNVYQARFEAVAWSATACHDGKKWMTIGVTGPDAKAVAVV
HYGGIPTDVaNSWAGDILRTQESSCTCIQGECFWVMTDGPANRQAQYRAFKAKQGKIVGQAEISFNGG
HIEECSCYPNEGKVECVCKDNWTGTNRPVLVISPDLSYRVGYLCAGLPSDTPRGEDSQFTGSCTSPMG
NQGYGVKGFGFRQGNDVWMGRTISRTSRSGFEILKVRNGWVQNSKEQIKRQVVVDNLNWSGYSGSFTL
PAELTKRNCLVPCFWVEMIRGNPEEKTIWTSSSSIVMCGVDHEIADWSWHDGAILPFDIDKM (Head sequen,c (E LO NO 8) hi one embodiment of this eighth aspect, the,:pOlypephdes are:
(a) at W4St 75%, 80%) 85%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally .20 occurring polypeptide includes 1, 2, 3,4, 5, 6, 7, 8, 9,10, or an II
011ie following stiniao acid residues relative to ID NO 8 when Ogned by protocof I or protocol 2::
(i) 408M, 101N, I 03AS, Ii U. 129Q, 160P, 16.1L, 63SITATIAll, 164V, 194Q, 201Y, 4421; or OD 101 N, 103A/$, 11 ID. 129Q; 160P, 161L, 163S/T/V/A/I, 164V, 194Q, 201Y, 44/1_ In :another embodiment, the pcilypeptides are (b) at: least 75%, 80%, 85%, 90%, 91%õ 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the nom-naturally OCturtitig polypeptide includes 2, 3, 4, 5, 6., 7,; 8,9,10. 11, 12, 13,,14õ Or all 15 of the.
following amino acid residne6=relative to SEQ ID NO:8 when aligned b-9 protocol I or p rotoCOI 2:
9$L, 408M, 101 N, :103A/S, 104V, I 1W, 129QA.4, 160P, 1611;.., 161,STr/WAIIIS17, 1:64V/P, 175I, 194Q, 201Y, 293t, 4421; or 01N, 103NS,104V, 1110, 1.29QN, 160P, 1.61L, lostrivwpwr, 164V/P, 1751, 194Q, 201Y, 2031, 4421 :vatiouflittizer embodimentS, the polypepti de nielude one or inote-of the following sets of arnina acid.tesidu6s relative to: SEQ. ID NO:8 whetualialied by ptotoCol 1. or protoda (i) 9811408M;
160P/163S;
101N/103A;
99C/162C;
99C/162C/172V;
129Q/442I;
99A/161L;
99A/161L/4421;
1611/4421;
129M/442I;
99A/129M/161L/4421/444A;
98L/99A/129M/161L/4421/444A;
129M/161L/4421/444A;
98L/129M/161L/4421/444A; and/or 1751/2031; or (ii) 160P/163S;
101N/103A;
99C/162C;
99C/162C/I72V;
129Q/442I;
99A/161L;
99A1161L/4421;

1.29M/4421;
30. 99A1129M/161114421/444A.:
98L/99A/129M/161L/4421/444A;
129M/161L/4421/444A;
9811129M/1611-144211444A; and/or 1751/2031.

Itt :various embodiments, the polypeptide includes 3.4,. 5.6, 7, 8, 9, or more of .the listed arthriO acid residues relative to SEQ ID 'NO;8 when aligned by protocol I Or protocol 2_ In a further embodiment., the polypeptideS cent:prises the amino acid sequence at least 75%,:
.80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid selected from the group consisting of N8 mutants liked in Table (SEQ ID
NO:35,38), when aligned by protocol 1, wherein the polypeptide includes all of the residues listed in Table I for an individual N8 mutant listed in Table In various further embodiments, the polypcptides are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%; 94%, 95%, 96%; 97%, 98%, Or 99% identical to the N8 NA
polypentide of SEQ ID NO;8, wherein the non-nanirally (*cuffing polypeptide incindos a 163E
amino acid mutation relative to SKI ID NO:a when aligned by protocol 1 , and further may optionally include a 194S Inutation relative to SEQ.ID.N0:8 when aligned by .protocol 1 or protocol In a ninth aspect the NA polypeptides are:
(a) at least 75%, 80.!'õ4, 85%, 90%,.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tO the N9 NA polypeptide of SEQ. ID .N0:9, and wherein the non-naturally occurring polypeptide includes 1,-2, 3, 4, 5,6, 7, 8,9, Or all 10 of the following airline acid residues relative to SEQ, ID NO.:9 When aligned by protocol .1 or protocol 2:
941', 95L., 100$, 120QN,160VINI, 101V, 191Q, 198Y, 439$, 441V; or (b) at least 75%, $0 ./.4 85%, 90%, 91% 92%, 93%, 94%, 95%, 90%, 97% 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally Oceniting polypeptide Mel tides 2., 3, 4, 5, 0, 7, 9,10, IL 12,13, Or all 14 of the following amino acid residues relative to: SEQ ID NO:9 when aligned by prOtocol 1 or protocol '2' 94Põ 95Lõ 98N, 100S/Aõ 126Q/M, 15217,1581, 160V/A/I/T, 16IV, 191Q, 198Y, 2001, 439S, 441V.
In this :ninth aSpectõ the N9 NA reference sequence is based on Nc,): reforoce :s0ajn:
.A,Arihu -YIK_R03912013 fromff7N9, teliFNOcncTSATAI IGAI AVL IGIANLGILNIGLHLKFGCNCSHS PETTN T SQ T I INNYYNETN I
T N
3() Ot...T.:E'R TST-ZNFNNIZKGLCT INSWHIYGKDNAVR I GE S S DVL'VTRE P'YVS CD
PDECRFYAL S QGTT I R
GKHSNGTIHDRSQYRALISWPLS S PP TVYN SRVEC IGWS S T SCHDGKSRMS Id I
SGPNNNASAVVWYN
RRPVAE INTWARN I L RTQE S ECVC HNGVC PVVFTDG S ATG PADTRI YYFKEGKILKWE S
LTGTAKH I E
EC SCYGERTG I TC TCRDNWQGSNRPVI QID PVAMTHTSQY I CS PVL TDNPRPND
PNIGKCNDPYPGNN
NNGVKGFSYLDGANTWLGRT I S TAS RS GYEMLKVPNALTDDRSK P I QGQT IVLNADWS GY S GS
FMDYW

AEGDCYRACPYVELIRGRPKEDIWWWTSNSIVSMCSSTEFLGQWWPDGAXIMPL J104d sequence is higniighted) (SEQ ID
in GUC embodiment of this ninth aspect, the polypeptides are:
(a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%., 95%, 96%, 97%, 98%, or 99% identical. to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide includes 1, 2, 3,4, 5, 6, 7, 8, 9, or all ID of the following wnino acid residues relgti7vc :to SP) ID N0:9 when aln4ned by protocol 1 or protocol 2:
94P, 951.õ 1()OS, 126Q/M, 160ViAll, 161V, 191O, 198Y, 439S, 441V, In another embodiment, the polypcptides are:
(b) atiCast: 75%; 80%; 85%, 90%; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide :includes 2, 3, 4, 5, 6,7, 8, 9, 10,11, .12, 13, or all 14 Of the following .amino acid residues relative to SEQ ID NO:9 When aligned by protocol I or protocol 2:
94P, 951.õ 98N, 100S/A, 126Q/1\4, 152T, 1581, 160V/All/T, 161.V, 191Q, 198Y, 2001, 439S, 44IV;
in various embodiments, the polypeptidesinelude 'Of MOM of the following! sets of amino acid tesidues relatiVe-M SEC). 10 NO:9 when aligned by proldeol 1 or prcitocol 2:
96C/1 59C;
96C/159C/441V:
96A/441A;
:96A/126M/4391/441A;
951,196A1126M/4391/44tA.
126M143911441A;
95L/126M/4391/441A;
94P/191T:
98N/100A; and/or 94P/191T/2001.
In various embodiments, the pelypeptideS include 3,4 5, 6, 7, 8, 9 or more of the listed amino acid residues relative to SEQ NO:9 when aligned by protocol 1 or protocol 2.
In a Maher embodiment, theipolypeptides: are Mat least 75%,:80%, 85%, 90%,:91%, 92%, 93%, 94%; 95%, 96%, 97%;: 98%, or 99% identical to the N9 NA polypeptide of :SEQ
NO:9, and wherein:the non-,naturally occurring polypeptide includes a 160Q
amino acid mutation relative to SEQ ID NO:9 When aligned by protocol 1, or may include a combination of 160Q/E and I.72V amino acid residues relative to SEQ ID NO:9 when aligned by protocol I or protocol 2.
In another embodiment, the disclosure provides composition, comprising one or more of the non-naturally occurring polypeptides of any embodiment or combination of embodiments disclosed herein linked to a scaffold. Linkage to scaffolds permits a plurality (2, 3,4, 3, 6, 7., 8.9, 10, or more) of the polypeptides to be displayed, which may enhance the immune response stimulated upon administration to a subject in need thereof, as described in the methods that follow. The compositions may comprise any scaffold suitable for an intended use. The one or more non-naturally occurring polypeptides may be linked 19 covalently or non-covalently to such a scaffold. In one embodiment, the scaffold comprises a protein scaffold; in this embodiment, the one or more non-naturally occurring polypeptides may be covalently linked to.the protein scaffold, including but not limited to by being expressed as a fusion protein with a protein component of the scaffold.
In another aspect the disclosure provides nucleic adds encoding the polypeptide or .15 fusion protein of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA
(such as an triRNA) or DNA in genomic or eDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
Such nucleic acid sequences may comprise additional sequences useful for promoting 20 expression andfor purification, of the encoded polypcptide, including but not limited to polyA
sequences, modified Kozak sequences, and sequences encoding cpitope tags, export signals, and secretory signals, nuclear localization signals, and plasma. membrane localization signals.
It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure.
25 In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence.
"Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product, 'Control sequences" operably linked to the nucleic acid sequences of the disclosure are nucleic acid 30 sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence.

Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid. sequences in a mammalian. system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, .EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, eedyso:ne, steroid-responsive).
The expression vector must be replicabie in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.
In another aspect, the disclosure provides host tells that comprise the nucleic acids, expression vectors (i.e: spisomal or ehromosomally integrated), non-naturally occurring polypeptides, fusion protein, or compositions disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the nucleic acids or expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polyeationie mediated-, or viral mediated transfection.
In another aspect, the present disclosure provides pharmaceutical compositions, comprising one or more polypeptides, fitSit)fi proteins, compositions, nucleic acids, expression vectors, and/or host cells of the disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the disclosure can be used, for example, in the methods Of the disclosure described below. The pharmaceutical composition may comprise in addition to the polypeptide of the disclosure (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer.
in some embodiments, the buffer is.a Tris buffer, a histidine buffer, a phosphate buffer., a Citrate buffer or an acetate buffer. 'Me pharmaceutical composition may also include a lyoproteetant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, tnethylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chloroeresol, phenylmereurie nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the phinniticeutital coMposition includes a surfactant eg,. polysorbate,20, pelyserbate-40, polysotbate- 60, polyxorbate65, pOlysorbate-80 polysorbate45...po1Otatner-188, sorbitan mOnOlautate, sorhitau inotioNlthitate., sorbitan inOnostearate, sOrbitaii inetiooleate,.SOrbitart trilaurate, sorbitan tristearate, sorbitan nioleaste, ora combination thereof, The pharmaceutical composition may also include a tonicity adjusting agent, .e.gõ
a compound that renders the formulation substantially isotonic or isoosmotic with humanblood.
Exemplaq: tonicity adjusting agents. include sucrose,. sorbitkil, .glyeine methioninc, manni tol, destreac, :inositol, sodium chloride, arena:tie and argininelsydroehloride. In other tanbodirrientS,. the pharmatetitiCal CoMpos iti on additionally includes a stabilizer. e:g;õ
nintecult *Ina. When combined:With a protein. Of interest substantially prevents Or reduces chemical and/or physical instability of the. protein of interest in lyophilized or liquid form..
Exemplary stabilizers: *el ode. sucrose, sorbing, glycine, inositol, sodium chloride.
Methioninc, arginineõ and argi nine hydrochloride.
Theip011ypeptideS, fusion proteins, Compositions,. nucleic. acids, .eXpression vectors, = and/or host delft may be the sole active agent in the pharmaceutical composition, or the composition or vaccine May further comprise One or More Other active agents.suitabie for an intended use.
The pOlypeptides, fusion:pa-Kt:ins. Compositions; pharina.ceu,tio.41 coMpositions, nucleic acids, expression vectors, andlor host .cells of the disclosure may be used fOr any .20 :suitable purpose, including; but not limited to treator limit.develOpment of influenza infections. For example, the potypeptides,..:frision proteins,..compositions,.pharinacentical compt,)sitions, nuelete..,acids, expression vectors., andlor hoStcells Maybe tiSed..to. elicit an Minim* response to influenza iru One type of immune response is a B-cell responSeõ
which results in the production ofatitibodieSagainst the antigen that elicited the immune response, While altantihodies are capable of.bhding to the antigen which elicited the immune response that resulted* antibody nroducfion, preferred umbodies are 'those that pros ide broad hcrerosubtypiepretection .against :influenza virus Thus,. the methods May elicit antibodies that bind, to an. influenza NA protein froth a virus selected from the group Om:slating:Of influenZa A viruses, influenza B OrtiseS, and. influenza. C
VirtOes, These 30. methods may then antibodies that bind to an influenza NA protein from an influenza virus selected from the .aroup ;consisting .of H1,112, H3., .I14,115, H6, .H7,118,119; Hi 0, 111 1. .H12, 1113., H14,1415, 1116,1-117. and HI 8. influenza A virus, andinfluenza. B
virus. The methods.
may elicit antibodies-that bind to an influenza NA protein from a *pin .ofinfluenzavirus selected from the group consisting of influenzatA/Californial07:12009 MIN I), 33.

A/Michigan/45/2015 (I-tINI), A/New Caledoniat20/1.999 (HI NI), ATWSNII933 A/Brevig Mis.sionil /1918 (H IN I), ANietriam,1203/2004 (1-15NI), A/Wisconsin/6712005 (H3N2), A/Swine/Missouri/212451412006 (H2N3), A/Red knot/Delaware Bay/31012016 (HI0N4), A/Shorebird/Delaware Bay/309/201 6 (H IONS), AlchickeniSiehuan/NCIPLI/2014 (115N6), A.Netherlands/219/2003 (117N7), Aaiangxi/IPB13b/2013 A/Anhui/I -Y1C...RG39/2013 (H7N9), .13/Phuket/3073/2013, B./Colorado/0612017 and antigenic variants thereof.
Protective antibodies elicited by methods of this disclosure can protect against viral infections by affecting any step in the life cycle of the virus. For example, protective antibodies may prevent, an influenza virus from attaching to a cell, entering a cell, releasing viral ribonuele.oproteins into the cytoplasm, forming new viral particles in the infected cell, and/or budding new viral particles from the infected host cell membrane.
Antibodies elicited.
by the methods of this disclosure preferably prevent influenza virus from attaching to or entering the host. cell, prevent fusion of viral membranes with endosomal membranes, or prevent release of newly formed virus from the infected host cell.
One aspect of this disclosure is a vaccine composition (vaccine) comprising any polypeptide, fusion protein, or composition disclosed herein, to protect subjects against infection by influenza virus.. Vaccine of this disclosure can also contain other components such as adjuvants, buffers and the like. Exemplary adjuvants include aluminum phosphate, .benzyalkonium chloride, ubenimex, and 021.; genetic adjuvants such as the IL-2 gene or fragments thereof, the granulocyte macrophage colony-stimulating factor (CiM-C'SF) gene or fragments thereof, the 1L-18 gene or fragments thereof, the ehe.mokine (C-C
motif) ligand.21 (CCL2I ).gene or fragments thereof, the 1L-6 gene or fragments thereof, CpG, LPS, TLR
agonists, and other immune stimulatory genes; protein adjuvants such 1L-2 or fragments thereof, the granulocyte macrophage colony-stimulating factor (GM-CSF) or fragments thereof, IL-18 or fragments thereof, the chemokine (C-C motif) ligand 21 (CCL21) or fragments thereof, 1L-6 or fragments thereof, CpG, LPS, TLR agonists and other immune stimulatory cytokines or fragments thereof; lipid adjuvants such as cationic liposomes, N3 (cationic lipid), imanophosphoryl lipid A (MPL1); other adjuvants including cholera. toxin, enterotoxin, Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine, marcaine, and levarnisole.
The vaccines of this disclosure may include immunogenic portions of more than one Type, Group, subtype, or strain of influenza virus. Such. vaccine may comprise nanoparticles, each of which comprises immunogenic portions from NA proteins from more than one Type, Group, subtype, or strain of influenza virus. Such a vaccine is'refened to as a multivalent .vaccine. A multivalent vaccine can comprise immunogenic portions from as many influenza NA proteins as necessary to elicit production of an immune response sufficient te protect 'against a desired breadth of virus Types, Groups, subtypes, or strains. In one embodiment, the vaccine comprises immunogenic portions of NA proteins from at least two different influenza strains (i.e., a bivalent vaccine), or from at least three different influenza strains (i.e., a trivalent vaccine), or from at least four di fibrent influenza. strains (i.e., a quadrivalent vaccine), or from at least five different influenza strains (i.e., a pentavalent vaccine). In one embodiment, the vaccine comprises immunogenic portions of NA proteins from at least six different influenza strains (Itexavalent).
This disclosure provides methods of vaccinating a subject against influenza virus, the method comprising administering a polypeptides, compositions, pharmaceutical compositions, nucleic acids, expression vectors, and/or host cells to the subject such that an immune response against influenza virus is produced in the subject.
.15 The subject may be any suitable subject, including but not limited to humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, seals, goats and horses; domestic mammals such as clogs and eats; laboratory animals including rodents such as mice, rats and guinea pigs; birds,.including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
in the vaccination methods of this disclosure, the subject being vaccinated may have been exposed to influenza virus. As used herein, the term "exposed" indicate the subject has come in contact with a person or animal that is known to be infected with an influenza virus.
Vaccines of this disclosure may be administered by any suitable technique, by means including, but not limited to; traditional syringes, needleless injection devices, or microprojectile bombardment gene guns. Suitable routes of administration include, but are not limited to, paremeral delivery, such as intramuscular, intradermal, subcutaneous, or intratnedullary injections, as well as intrathecal, direct intraventrieular, intravenous, intraperitoneal, intranasal, or intraocular injection.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.

Examples We provide sequences of recombinant NA proteins in Which head domains comprising stabilizing mutations are connected to tetrarnerization domains. We initially found that many wild-type sequences of beta propeller head domains from certain NA
subtypes adopted "open" conformations in which the head domains extended individually off of the stalk-like tetrainerization domain, without fomiing the crystallographically observed "closed" tetromer. Constructs comprising the head domains from other NA
subtypes formed closed tetramers naturally. Similar constructs from yet other stilay.pes tbrined mixtures of open and closed tetramers. We identified Specific mutations at multiple locations in NA
sequences that dictate the open or closed conformational state of NA
tetramers, and used these mutations to generate closed, stabilized tetramers from multiple NA
subtypes. We also converted a naturally closed N.A tetramer to a fully open conformation by substituting residues that we identified as pivotal for tetramer closure, confirming the importance of these residues for determining the conformational state of NA. M.onoelonal antibodies (inAbs) that bind across the interface of two neighboring protomers in the closed configuration bind better to closed tetratners than open tetramers. The mutations we provide may be useful for stabilizing other NA proteins that naturally form open tetramers when produced recombinantly.
Together, the disclosure provides mutations at defined locations in NA
proteins that close the open structures of various NA tetramers. These stabilized NAstructures can be used as vaccine antigens in either soluble form or when presented on scaffolds.
Materials and Methods Protein design and expression NA constructs were expressed by transient transfecdon in Expi293F cells (ThermoFisher Scientific) at a density of 2.5x 10A6 cells/ml using ExpiFectaminerm.293 Transfection Kit (Thermo Fisher Scientific). The supernatants were harvested 5 days post transfection and centrifuged at 4000 rpm to remove cell debris. The culture supernatants were sterile filtered prior to purification by immobilized metal affinity chromatography (MAC).
Clarified supernatant was incubated for 2 h at room temperature with Ni ScpharoseTM High Performance histidine-tagged protein purification resin (GE Healthcare) and separated through affinity chromatography. Bound protein was eluted with 300 niM
imidazole, 50 rirM
Tris-HCI and 0.5 M Naa. Eluted protein was further purified by size exclusion 'chromatography two phosphate-buffered saline (PBS) using a Superelei" 200 Increase 10/300 column (ciE Life Sciences).
NA antigenic characterization Several. mAbs are known that can be used to assess the antigenieity or conformational state of NA. We used multiple mAbs for this purpose, including CD6, a mAb that binds across the interface of two prototners in the closed, crystallographically observed C4-symmetric eonfiguratien (Wan et al., Md. (.7ornms, 6:6114). We found that.CD6 bound better to recombinant NA proteins: that formed closed tetramers than NA proteins that formed open tetramers.
A forteBio 'Octet"114TX instrument was used to measure binding of NA. proteins to antibodies that target several antigenic sites. All assays were performed in PBS supplemented with 1% bovine serum albumin (BSA) to minimize nonspecific interactions. The final .volume for all solutions was 50 pltwell. Assays were performed at 30 '"C`. in solid black 384-well plates. NA was loaded for 300-600 s on HIS1K tips, which were then dipped to capture mAbs for 600 s. mAbs were then allowed to dissociate for 300-600 s in PBS + 1%
BSA. Data analysis was carried out using Octet software, version II. High capture levels of protein (same as reference proteins or higher) were part of the selection process for EM analysis.
Binding of mAb to protein was the second step of the selection process for EM
analysis.
NA activity assays Neuraminidase activity was measured with the NA-Fluor Influenza Neuraminidase Assay Kit according to the manutlicturer's protocol. Briefly, 50-100 airtml of protein was used as a start concentration and 2-fold dilutions were prepared in duplicate in a black 96-well, flat bottom plate for each protein sample. The wells in column 12 were left empty for controls. NA-Fluor Substrate was prepared according to the protocol and added to each well.
Plates were incubated for 1 h at 37 'T. and reactions were stopped with NA-Fluor Stop Solution. Plates were read using an excitation wavelength range of 350 rim to 365 Mil and an emission wavelength range of 440 nm to 460 nm. Background control wells were subtracted for each protein serial dilution. Finally, protein dilutions wereplotted versus relative fluorescence unit (RFU) values.

EM sample preparation We used negative stain electron microscopy and particle averaging to assess whether the head domain of recombinant NA proteins adopted the open or dosed structure. Hiram 2 shows representative examples of two-dimensional class averages of recombinant NA
proteins that fomt open tetramers (NIcataa., Ni Nras, Ni Ni WSN3.1 and N6skimani4), mixtures of open and closed tetramers (1S7i.1.03, N831)11, and N9Aamaa), and dosed tetramers (N2w1os, .N3swta..~owi, .N4sta kootVelawatv16, and N5shorehird,ndew;tret6).
Proteins were diluted to a concentration of about 0.02 mg/ml with buffer containing mM HEPES, pH 7.0õ. and 150 mM NaCI and adsorbed to a glow-discharged carbon-coated 10 copper grid. The grid was washed with a drop of the same buffer three times and, stained with 0.75% uranyl formate. Images were recorded at a nominal magnification of 100,000 (pixel size: 0.22 nm) using SerialEM. software onan Menai 120 electron microscope equipped with an FEI Eagle CO) camera and operated at 200 kV. Particles were selected from the micrographs automatically using in-house software (Yaroslav Tsybovsky, unpublished) and .15 extracted into .128x128-pixe1 boxes. Reference-free 21) classification was performed using Relion 1.4.
Results Referring to Figure 1, three different recombinant NA designs were expressed (Figure IA), purified by IMAC, and examined by size exclusion chromatography (SEC). In the first recombinant NA configuration, an N-terminal &His tau was appended to residues 35-469 of A/California/0712009 (HMI) NA (SW ID NO: 0. comprising the native stalk and head domains. A second recombinant NA configuration was tested in which the cyto.solic, transmembrane, and stalk domains of the wild-type NA (residues 1-77 in SEQ ID
NO: 1) were replaced by a 6xHis-tag, an hVSAP domain to drive protein tetrainerization (Xu et al., .1.
Vim!. 82:10493-10501), a thrombin cleavage site, and a two-residue Gly-Gly linker. A third recombinant NA configuration was tested in which the cytosolic, minsmembrane, and stalk domains Of the wild-type NA (residues 1-82 in SW m NO: 1.) was replaced by a 6x.flis-tag, an hVSAP domain, a thrombin cleavage site, and a two-residue Gly-Gly linker.
Recombinant NAs in which the head domain started at position 83 showed homogeneous SECprofiles (Figure 1B) with a maior peak corresponding to the estimated molecular weight of NA
tetramers, with minimal aggregation. Subsequent constructs were designed with head domains starting at position 83.

Referring to Figure 2, purified recombinant NA proteins from a number of subtypes were characterized structurally by negative stain ENE Representative NM from the N2, N3, N4, N5 subtypes formed closed tetramerie structures in which the head domain resembled. the C4-symmetric structure classically observed by X-ray crystallography.
Representative NM
from the Ni and N6 subtypes formed open tetramers in which the head domains did not form a single, compact structure. Representative NM from the N7, N8, and N9 subtypes formed mixtures of open and closed tetramers.
Referring to Figure 3, in one non-limiting example, we designed a series of recombinant NA Mutants based on the wild-type A/California/0712009 (11 IN 1) NA sequence that resulted in a protein that formed a closed tetramer. Introducing ten mutations into AICa1:ifornia/0712009 (MINI) NA (construct name 94_N 1 -Ca109,...danfav2 in Table I; SEQ
ID NO:79) resulted in 45% closed tetramers. Additional mutations and a reverse mutation result in a protein (construct name 155...N1 -Ca1119-el 30...T453V in Table 1;
SEQ ID NO:! 3) that forms 90-100% closed recombinant NA tetramers. The 991), 1771, 1961' and mutations provide improved hydrophobic packing at the inter-protomeric interface. The 1.65S
mutation helps stabilize the closed conformation of a loop that participates in the inter-protomeric interface. The 161V and I nA mutations remove a eysteinc and optimize packing, which improves expression. The 100L, 408M and 419V mutations improve expression by removing polar residues in a region of the protein that is partially hidden from solvent.
Referring to Figure-4, in another non-limiting example, stabilizing mutations introduced into the sequence of A/Michigan/45/2015 (Hi Ni) NA, which forms an open tetramer When the wild-type sequence for the head domain is used, result in a prOtein (constrtiet name 1.74...N1-Mi1.5..e155...T131Q in Table 1; SEQ ID NO:1.8) that forms 100%
closed tetramers. The 131Q mutation optimizes. packing and helps stabilize the dosed conformation of a loop that participates in the inter-protomeric interface.
Referring to Figure 5, in another non-limiting example, stabilizing mutations introduced into the sequence of AMSN/1933 (11INI) NA, which forms an open tetramer when the wild-type sequence for the head domain is 'used, result in a protein (construct name :366N1-WSN33c155(3105SJI.06V_A 57T_V1631._A I 66V_R2100 in Table 1; SEQ ID
NO:43) that forms 80% closed tetramers. The 57T and 166V mutations provide improved hydrophobic packing at the inter-protomerie interface. The 210G mutation removes electrostatic repulsion at the inter-protoinerie interface. The 1055, 106V and 1631 mutations help optimize packing to stabilize. the closed conformation of a loop that participates in the inter-protomeric interface.

Referring to Figure 6, in another non-limiting example, stabilizing mutations introduced into the sequence of A/Vietnam/1203/04 (H5N1) NA, which forms an open tetramer when 10 Stabilizing mutations originally identified in A/California107/2009 (HINI) NA are used, result in a protein bearing 14 mutations (construct name 3.54_NI-VN04 c155_1106V J131Q.V163LA166V in Table 1; SEQ ID NO:40) that forms 100%
dosed tetramers. The 106V, 131Q and 1631 mutations help optimize packing to stabilize the closed conformation of a loop that participates in the inter-protometie interface. The 166V
mutation provides improved hydrophobic packing at the inter-protomeric interface.
Referring to Figure.7; two mutations introduced into the recombinant A/Jiangxi-Donghui346-2/2013 (H IONS) N8 NA (construct name 28.5..N8-hangxi-Donghti2OI
3...E162P-Q.I65S in Table 1; SEQ ID NO:36) resulted in the formation of 100% closed tetramers. The 162P and 165S mutation help stabilize the closed conformation of a loop that participates in the inter-protornerie interface.
Referring to Figure 8, we introduced substitutions at positions that are critical for 'stabilizing the closed tetrameric structure of NA into a naturally closed recombinant NA to open up the closed, compact structure. Three mutations at positions that had been identified to stabilize open tetmmers resulted in the formation of exclusively open tetnuners of the NA
from A/Wisconsin/67/2005 (H3N2) (construct name 255. JN2-Wis05V165Q
1.176KJ195.5 in Table 1; SEQ ID NO:34). The 176V and 195S mutations decrease the amount of inter-protomeric hydrophobic packing that is natively present. The 165Q mutation allows for increased flexibility of-a loop that participates in the inter-protomerie interface.
Referring to Figure 9, a Basic Local Ahgnment Search Tool protein (BLASTp) alignment between reference sequences and any arbitrary sequence of the same subtype allows for identification of positions for the described mutations in any NA
sequence.
Sequence positions in arbitrary 'NA sequences are identified based on alignment to corresponding positions in reference sequences. Many sequence alignment tools are known to those of skill in the art and can be used to align arbitrary NA sequences to the provided reference sequences. Here we provide protocols for aliening sequences using the. online BLASTp tool provided by the National Institute of Health (protocol 1) as well as a standalone executable version of the BLAST software that can be run locally on any computer (protocol 2).
Overall, we identified mutations that result in recombinant NA proteins that adopt closed, C4-symmetric,. . or open, non-symmetrie conformations. Mutations that fillcavities in NA are helpfid for tetramer closure. In some non-limiting examples, engineered disulfide bonds are helpful for tetrarner closure. Certain amino acid positions, including but not limited to position 165, appear most relevant for dictating the open or closed conformational state of NA tetramers. In addition, other mutations substantially improve overall protein expression levels.
Table 1 Mutations Mutations highlighted Name Sequence included in uppercase Closure VKLAGNSSLCPVSGWAPLSKDN
vkiagnss1cpvsgwaPLsken SVRIGSKGEVEVIREPFISCS?
svrigskgEvfvirepfiscsp LECRTFFTWALLNDKHSNGT
leertffitqga1lndkhsngt IKDRSPYRTLMSCPIGSVPSPS
ikdrspyrt1mscpigSvpspS
NSRFESVAWSASACHDGINWLT
nsrtesvawsasachdginwit IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd Ni TIKSWRNNILRTQESECACVNG
tikswrnniIrtqesecacvng numbering:
SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsnggasykifri 94 Ni- EKGKIVKSVEMNAPNYHYEECS
ekgkivksvemnapnyhyeecs 51, Ca109 d CYPDSSFITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp 45%
anfav2 WVSENQNLEYQIGYICSGIEGD 113E/170S, wvsfnqn1eygigyicsgifgd 165S, NPRPNEKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr 19V, 453T
NGFFMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHBEL
gdivginewsgysgsfvMhpe1 TCLDCIVPCFWVELIRGRPKEN
tg1dciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTTGWS
tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:79) NO:79) VKLAGNSSLCPVSGWAPYSKDN
vkLagnss1cpvsgwaPyskdn SVRIGSKGEVEVIREPFISCS?
svrigskgEvfvirepfiscsp LECRTFFLTQGALLNDKHSNGT
lecrtffltqga1lndkhsngt IKDRSPYRTLMSCPIGSVPSPS
ikdrspyrt1mscpigSvpspS
NSRFESVAWSASACHDGINWLT
nsrfesvawsasachdginwit IGITGPDNGAVAILKYNGIITD
igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG tikswrnni1rtgesecacvng Ni 112 Ni- SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsnggasykifri Cal09 d EKGKIVKSVEMNAPNYHYEECS numbering:
99P/196T/20 ekgkivksvemnapnyhyeecs anfav2 CYPDSS'EITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp 40%
no- WVSENQNLEYQIGYICSGIEGD 51, wvsfnqn1eygigyicsgifgd 113E/170S, space-B NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv 165S, 453T
KGFSFEYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik ODIVGINEWSGYSGSFVOHBEL
cidivginewsgysgsfvqhpe1 TOLDCIRPCFWVELIRCRPKEN
tg1dcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTTGWS
tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:80) NO:80) VKLAGNSSLCPVSGWAPLSKDN vkLagnss1cpvsgwaPLskdn Ni SVRIGSKGEVEVIREPFISCS?
svrigskgEvfvireptiscsp numbering:
lecrtffit LECRTFELTQGALLNDKHSNGT eiga1lndkhsngt 113 Ni- IKDRSPYRTLMSCPIGSVPSPT
ikdrspyrt1mscpigSvpspT
Cal09 D NSRFESIAWSASACHDGINWLT 6T/205I, nsrfesIawsasachdginwit 80-90%
113E/170T, igiTgpdngavaIlkyngiitd 165S, TIKSWRNNILRTQESECACVNG
tikswrnnilrtgesecacvng IDOL/4.08MP"
SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsnggasykifri 19V, 4531 EKGKIVKSVEMNAPNYHYEECS
ekgkivksvemnapnyhyeecs SUBSTITUTE SHEET (RULE 26) CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp WVSENQNLEYQIGYICSGIFGD
wysfngnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgfsfkygnqvwiqrtksissr NGFEMIWDPNGWTGIDNNESIK
ngtemiwdpngwtgtdnntsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfyMhpe1 TGLDCIVPCFWVELIRGRPKEN
tg1dciVpctwvelirgrpken TIWTSGSSISFOGVNSDITGWS
tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:81) NO:81) VKLAGNSSLCPVSGWAPLSKDN
vk1Lagnss1cpvsgwaPLskdn SVRIGSKGEVFVIREPFISCS?
svrigskgEvfvirepfiscsp LECRTFFLIQGALLNDKHSNGT
lecrtff1tqga11ndkhsngt IKDRSPYRTLMSCPIGSVPSPS
ikdrspyrt1mscpigSvpspS
NSRFESIAWSASACHDGINWLT
nsrfesIawsasachdginwit IGITGETNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng numbering:
114 Ni-SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsngqasykifri Ca109 D EKGKIVKSVEMNAPNYHYEECS
6T/205I, ekgkivksvemnapnyhyeecs F2TI y1 CYPDSSEITCVCRDNWHGSNR? 113E/170S
cypdsseitcycrdnwhqsnrp 100%

WVSFNQN= , wysfngnleyqigyicsgifgd , NPRPNDKTGSCGPVSSNGANGV 100L/408M/4 nprpndktgscgpvssngangv kgfsfkygngvwigrtksissr 9V, NOFEMIWDETGWTGIDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfyMhpe1 TGLDCIVPCFWVELIRGRPKEN
tg1dc1Vpcfwvelirgrpken TIWTSGSSISFCGVNSDITGWS
tiwtsgssisfcgynsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pttidk (SEQ ID
NO:82) NO:82) VKLAGNSSLCPVSGWAPYSKDN
vklagnss1cpvsgwaPyskcin SVRIGSKGEVFVIREPFISCS?
svrigskgEvfvirepfiscsp LECRIFFLIQGALLNDKHSNGT
lecrttfltqga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPT
ikdrspyrt1msVpigSvpspT
NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD
igiTgpdngayaIlkyngiitd TIKSWRNNILRTQESECACVNG Ni tlkswrnnlirtgesecacvng 127 N1- SCFTVMTDGPSNGQASYKIFRI numbering:
softvmtdgpsngqasykifri Ca109 D EKGKIVKSVEMNAPNYHYEECS 99P/177I/19 ekgkivksvemnapnyhyeecs 22TI_Cy CYDDSSEITCVCRDNWHGSNR? 6T/205I, cypdsseitcycrdnwhgsnrp 30-40%
sK0 no- WVSFNQN=QIGYICSGIFGD 113E/1701, wvsfnqnleyqigyicsgifgd space-B NPRPNDKTGSCGPVSSNGANGV 165S, 4531, nprpndktgscgpvssngangv kgfsfkygngywigrtksissr ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvqhpe1 TGLDCIRPCFWVELIRGRPKEN
tg1dcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTTGWS
tiwtsgssisfcgynsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:83) NO:83) VKLAGNSSLCPVSGWAPLSKDN
vklagnss1cpvsgwaPLskdn SVRIGSKGEVFVIREPFISCS?
syrigskgEvfyirepfiscsp LECRTFFLIQGALLNDKHSNGT Ni lecrtffltqga1lndkhsngt IKDRSPYRTLMSVPIGSVPSPT numbering: ikdrspyrt1msVpigSvpspT
128 Ni- NARFESIAWSASACHDGINWLT 99P/177I/19 nArfesIawsasachdginwlt Cal09 D IGITG2DNGAVAILKYNGIITD 6T/2 051, igiTgpdngayaIlkyngiitd F2TI Cy TIKSWRNNILRTQESECACVNG 113E/1701, tikswrnni1rtgesecacvng 90-100%
sKO_T45 SCFTVMTDGPSNGQASYKIFRI 165S, scftvmtdgpsncidasykifri ekgkivksvemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR? 19V, cypdsseitcycrdnwhgsnrp WVSENQN=EYQIGYICSGIEGD 161V/172A
wvstngnleyqigyicsgitgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgfsfkygngywigrtksissr SUBSTITUTE SHEET (RULE 26) NCFEMIWDPNGWTGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpe1 TGIDCIVPCFWVELIRGRBKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:84) NO:84) VKLAGNSSLCPVSGWAPLSKDN
vklagnssicpvsgwaPIskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFITQGALLNDKHSNGT
lecrtifitqga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrtImsVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwlt IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd TIKSWRNNIIRTQESECACVNG
tikswrnni1rtgesecacvng 130 N1- numbering:
Cal139 D SCFTVMTDGPSNGQASYKIFRI 9 scftvmtdgpsngqasykitri F2TI Cy EKGKIVKSVEMNAPNYHYEECS
6T/2051, ekgkivksvemnapnyhyeecs sK0 Ell CYPDSSEITCVCRDNWHGSNR? 1653, cypdsseitcvcrdnwhgsnrp 90-100%

100L/408M/4 wysfnqnleycligyicsgifgd NPRPNDKTGSCGPVSSNGANGV 19V 161V/172A, 453T
nprpndktgscgpvssngangv , KGFSFKYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpctwvelirgrpken TIWTSGSSISFCGVNSDTTGWS
tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgaeipftidk (SEQ ID
NO:85) NO:85) VKLAGNSSLCPVSGWAPYSKDN
vklagnssicpvsgwaPyskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFITQGALLNDKHSNGT
lecrtffltqgatlndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwlt INITCPDNGAVAILKYNGIITD
igiTgpdngavaIlkyngiitd tikswrnni1rtqesecacvng Cai09 D SCFTVMTDGPSNGQASYKIFRI numberi scftvmtdgpsngqasykitri F2TI_Cy EKGKIVKSVEMNAPNYHYEECS
99P/14,19 ekgkivksvemnapnyhyeecs sK0 no- CYPDSSEITCVCRDNWHCSNRP 6T/2051 cypdsseitcvcrdnwhgsnrp 90-100%
, space- WVSFNQNLEYQIGYICSGIFGD
wvsfilqnleyuligyiusgifd 1655, 453T, nprpndktgscgpvssngangv kgtstkygngvwigrtksissr NGFEMIWDRNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvqhpe1 TGLDCIRPCFWVELIRGRPKEN
tgldcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTTGWS
tiwtsgssistcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:86) NO:86) VKLAGNSSLCPVSGWAPLSKDN
vklagnssicpvsgwaPIskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFITQGALLNDKHSNGT
lecrtff1tqga11ndkhsngt IKDRSPYRTLMSVPIGEVPSPY
ikdrspyrttmsVpigevpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwlt 134 N1- ICITGPDNGAVAILKYNGIITD Ni igiTgpdngavaIlkyngiitd Ca109 D TIKSWRNNILRTQESECACVNG numbering:
tikswrnni1rtqesecacvng F2TI Cy SCFTVMTDGPSNGQASYKIFRI 99P/1771/19 scftvintdgpsngclasykifri sK0 S16 EKGKIVKSVEMNAPNYHYEECS 6T/205I, ekgkivksvemnapnyhyeecs 50%
5E_T453 CYPDSSEITCVCRDNWHGSNR? 100L/4 08M/4 cypdsseitcvcrdnwhgsnrp V Ell0D WVSFNQNLEYQIGYICSGIFGD 19V, wvsfnqnleyqigyicsgifgd T170Y NPRPNDKTGSCGPVSSNGANGV 161V/1727%
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
qdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws SUBSTITUTE SHEET (RULE 26) WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:10) NO:10) vk1agnsslcpvsgwa2Lskcin SVRIGSKGEVFVIREPFISCS? svrigskgEvfvirepfiscsp LECRTFFLTQGALLNDKHSNGT lecrtifitqgallndkhsngt IKDRSPYRTLMSCPIGSVPSPT ikdrspyrtimscpigSvpspT
NSRFESIAWSASACHDGINWLT nsrfesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD igiTgpdsgavaIlkyngiitd Ni TIKSWRNNILRTQESECACVNG tikswrnni1rtqesecacvng numbering:
SOFTIMIDGPSDGQASYKIFRI 99P/1771/19 scttimtdgpsdgqasykitri 140 N1- EKGKIIKSVEMKAPNYHYEECS ekgkiiksvemkapnyhyeecs 6T/205I, Mil5 DF CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp 10-20%
113E/170T, 2TI WVSFNQN1,EYQMGYICSGVFGD wvsfnqnleyqmgyicsgvfgd 165S, NPRPNDKTGSCGPVSSNGANGV
100L/4 08M/4 nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr 19V, 453T
KGFEMIWDPNGWTGTDNKFSIK kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTTGWS tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:11) NO:11) VKLAGNSSLCPVSGWAPYSKDN vklagnsslcpvsgwaPyskdn SVRIGSKGEVFVIREPFISCS? svrigskgEvfvirepfiscsp LECRTFFITQGALLNDKHSNGT lecrtffltqgatlndkhsngt IKDRSPYRTLMSCPIGSVPSPT ikdrspyrtimscpigSvpspT
NSRFESIAWSASACHDGINWLT nsrfesIawsasachdqinwlt IGITGPDSGAVAILKYNGIITD igiTgpdsgavaIlkyngiitd TIKSWRNNILPTQESECACVNG tikswrnni1rtqesecacvng Ni SOFTIMIDGPSDGQASYKIFRI
scftimtdgpsdgciasykifri 141 Ni- nu mbering:
EKGKIIKSVEMKAPNYHYEECS Mil5 DF 99P/1771/19 ekgkiiksvemkapnyhyeecs CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp 40%
2TI no- 6T/205I, WVSFNQNIEYQMGYICSGVFGD wysfnqnleyqmgyicsgvfgd space-B 113E/170T, NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv 165S, 453T
KGFSFKYGNGVWIGRTKSISSR kgtstkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVQHFEL qdivginewsgysgsfvqhpe1 TGLDCIRPCFWVELIRGRPEEN tgldcirpctwvelirgrpeen TIWTSGSSISFCGVNSDTTGWS tiwtsgssistcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:12) NO:12) VKLAGNSSLCPVSGWAPLSKDN vklagnsslcpvsgwaPLskdn SVRIGSKCDVFVIREPFISCS? svrigskgdvfvirepfiscsp LECRTFELTWALLNDKHSNGT lecrtff1tqgailndkhsngt IKDRSPYRTLMSVPIGSVPSPY ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT nArfesIawsasachdginwlt IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd Ni TIKSWRNNILRTQESECACVNG tikswrnniirtqesecacvng numbering:
SCFTVMTDGPSNGQASYKIFRI scftvmtdgpsngqasykifri Ca109- EKGKIVKSVEMNAPNYHYEECS
6T/205I, ekgkivksvemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhqsnrp 90-100%
c130 T4 165S, WVSFNQNLEYQIGYICSGIFGD

wvsfnqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv 19V, KGFSFKYGNGVWIGRTKSISSR 161V/172A kgfsfkygngvwigrtksissr NGFEMIWDDNGWIGTDNNFSIK ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL qdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NU:13) NU:13) 300 Mil VKLAGNSSLCPVSGWAPLSKDN Ni vklagnsslcpvsgwaPLskdn 5_c155 SVRIGSKGDVFVIREPFISCS? numbering: svrigskgdvfvirepfiscsp SUBSTITUTE SHEET (RULE 26) LECRTFFITQGALLNDKHSNGT 99P/177I/19 1ecrtffltqqa1lndkhsnqt IKDRSPYRTLMSVPIGSVPSPY 6T/205I, ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT 1655, nArfesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD 100L/4 08M/4 igiTgpdsgavaI1kyngiitd TIKSWRNNILRTQESECACVNG 19V, tikswrnni1rtgesecacvng SCETIMTDGPSDGQASYKIFRI 161V/172A scftimtdgpsdgclasykifri EKGKIIKSVEMKAPNYHYEECS
ekgkiiksvemkapnyhyeecs CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp WVSENQNLEYQMGYICSGVFGD
wvsfnqnleyqmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:14) NO:11) VKLAGNSSLCPVSGWAPLSKNN
vklagnsslcpvsgwaPLskEn AVRIGSKGDVFVIREPFISCS?
Avrigskgdvfvirepfiscsp LECRTFFITQGALLNDKHSNGT
lecrtff1tqga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtqesecacvng SCETIMIDGPSDGQASYKIFRI 99P/177I/19 seftimtdgpsdgqasykifri Mil cl EKGKIIKSVEMKAPNYHYEECS 6T/205I, ekgkiiksvemkapnyhyeecs 55 D103 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 80-90%
N S105A WVSENQNLEYQMGYICSGVFGD 100L/408M/4 wvsfnqnleyqmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktqscgpvssnganqv KGFSFKYGNGVWIGRTKSISSR 161V/172A, kgfsfkygngvwigrtksissr kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TOLDCIVPCFWVELIRGRPEEN
tqldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:15) NO:15) VKLAGNS SLCPVS GWAPL SKDN
vklagnss1cpvsgwaPLskdn SVRIGSKGDIFVIREPFISCSP
svrigskgdItvireptiscsp LECRTFFITQGALLNDKHSNGT
lecrtfr1tqqa11ndkhsngt IKDRSPYRTLMSVPIGSPPSPY
ikdrspyrt1msVpigSPpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtqesecacvng SCFTIMTDGPSDGQASYKIFRI 99P/177I/19 scftimtdgpsdggasykifri 165¨N1-Mil5 cl EKGKIIKSVEMKAPNYHYEECS 6T/205I, ekgkiiksvemkapnyhyeecs 55 V114 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 100%
I V166P WVSENQNLEYQMGYICSGVEGD 100L/408M/4 wvsfnqnleyqmgyicsgvfgd NPRETDKTGSCGEWSSNGANGV i9V, nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR 161V/172A, kgfsfkygngvwigrtksissr KGFEMIWDPNGWIGTDNKFSIK 114I/166?
kgtemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pttidk (SEQ ID
NO:16) NO:16) 167 Ni-VKLAGNSSLCPVSGWAPLCKDN Ni vklagnsslcpvsgwaPLCkdn Mil5 cl SVRIGSKGDVFVIREPFVSCS? numbering:
svrigskgdvfvirepfVscsp LECRTFFITQGALLNDKHSNGT 99P/177I/19 lecrtifitqqa11ndkhsngt 100%
55¨S101 IKDRSPYRTLMSVPICSVPSPY 6T/205I, ikdrspyrt1msVpiCSvpspy NARVESIAWSASACHDGINWLT 1653, nArVesIawsasachdginwlt G164C¨ IGITGPDSGAVAILKYNGIITD 100L/408M/4 igiTgpdsgavallkyngiitd SUBSTITUTE SHEET (RULE 26) F174V S TIKSWRNNILRTUSECACVNG 19V, tikswrnni1rtgesecacvng 444V SCETIMTDGPSDGQASYKIFRI 161V/172A, scftimtdgpsdggasykifri EKGKIIKSVEMKAPNYHYEECS 101C/122V/1 ekgkiiksvemkapnyhyeecs CYPDSSEITCVCRDNWHGSNR? 64C/174V/44 cypdsseitcvcrdnwhgsnrp wvsfnqnleyqmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgtstKygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSSIVFCGVNSDTVGWS
tiwtsgssiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:17) NO:17) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGDVEVIREPFISCS?
svrigskgdvfvirepfiscsp LECRQFFITQGALLNDKHSNGT
lecrQffitgga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtgesecacvng SCFTIMTDGPSDGQASYKIFRI 99P/1771/19 scftimtdgpsdggasykifri Mil cl EKGKIIKSVEMKAPNYHYEECS 6T/205I, ekgkiiksvemkapnyhyeecs 55 T131 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 100%
WVSFNQN=QMGYICSGVFGD 100L/408M/4 wvsfngnleygmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv KGESYKYGNGVWIGRTKSISSR 161V/172A, kgfsfkygngvwigrtksissr kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN
tgldciVpdfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (3E0 ID wpdgae1pftidk (SEQ ID
NO:10) NO:10) VKLAGNSSLCPVSGWAPLSKNN
vklagnsslcpvsgwaPLskNn AVRIGSKGDVFVIREPFISCS?
Avrigskgdvtvireptiscsp LECRQFFITQGALLNDKHSNCT
lecrQffltgga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESTAWSASACHDGINWLT nArtesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD
igiTgpdsgavaIlkyngiitd numbering:
TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng 175 Ni- SCETIMTDGPSDGQASYKIFRI
scftimtdgpsdggasykifri Mil5 cl EKGKIIKSVEMKAPNYHYEECS 611205"
ekgkiiksvemkapnyhyeecs 55 D103 CYPDSSEITCVCRDNWHGSNR? 165S, 100L/408M/4 cypdsseitcvcrdnwhgsnrp 90-100%
N S105A WVSENQN=QMGYICSGVFGD
wvsfngnleygmgyicsgvfgd 19V, nprpndktgscgpvssngangv , kgfsfkygngvwigrtksissr 131Q , KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN
tg1dciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaeIpttidk (SEQ ID
NO:19) NO:19) VKLAGNSSLCPVSGWAPLSKDN Ni vklagnsslcpvsgwaPLskdn SVRIGSKGDIFVIREPFISCS? numbering: svrigskgdIfvirepfiscsp LECRQFFITQGALLNDKHSNGT 99D/1771/19 lecrQffitgga11ndkhsngt 176¨N1- IKDRSPYRTLMSVPIGSPPSPY 6T/205I, ikdrspyrt1msVpigSPpspy Mil5¨cl NARFESIAWSASACHDGINWLT 1653, nArfesIawsasachdginwlt 55 V114 I V166P IGITGPDSGAVAILKYNGIITD 100L/408M/4 igiTgpdsgavaIlkyngiitd 100%
T131Q TIKSWRNNILRTQESECACVNG 19V, tikswrnni1rtgesecacvng SCFTIMIDGPSDGQASYKIFRI 161V/172A, scftimtdgpsdggasykifri EKGKIIKSVEMKAPNYHYEECS 131Q, ekgkiiksvemkapnyhyeecs CYPDSSEITCVCRDNWHGSNR? 114I/166P
cypdsseitcvcrdnwhgsnrp SUBSTITUTE SHEET (RULE 26) WVSENQNLEYQMGYICSGVEGD wvsfngnleygmgyicsgvfqd NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv KGES7KYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS tiwtsgssistcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:20) NO:20) VKLAGNSSLCPVSGWAPLSKDN vklagnsslcpvsgwaPLskdn SVRIGSKGDVEVIREPFISCS? svrigskgdvfvirepfiscsp LECRQFFITQGALLNDKHSNGT lecrQffltggallndkhsngt IKDRSPYRTLMSVPIGSVPSPY ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT nArfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtgesecacvng SCFTIMTDGPSDGQASYKIFRI 99P/177I/19 scftimtdgpsdggasykifri Ni15 cl EKGKIIKSVEMKAPNYHYEECS 6T/205I, ekgkiiksvemkapnyhyeecs 55 T131 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 90-100%
Q S442I WVSENQNLEYQMGYICSGVEGD 100L/408M/4 wvsfncinleygmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR 161V/112A, kgtstrcygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK 131Q/4 42I kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN tgldciVpcfwvelirgrpeen TIWTSGSIISFCGVNSDTVGWS tiwtsgsIisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:21) NO:211 VKLAGNSSLCPVSGWAPLSKDN vklagnsslcpvsgwaPLskdn SVRIGSKGDVFVIREPFISCS? svrigskgdvfvirepfiscsp LECRMFFITQCALLNDKHSNGT lecrMff1tqga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT nArfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtgesecacvng SCETIMTDGPSDGQASYKIERI 99P/177I/19 scftimtdgpsdgulasykifLi 181¨N1-M115 cl EKGKIIKSVEMKAPNYHYEECS 6T/205I, ekgkiiksvemkapnyhyeecs 55 T31 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 90-100%
WVSENQNLEYQMGYICSGVEGD 100L/408M/4 wvsfncinleygmgyicsgvfqd M¨S442I NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv KGES7KYGNGVWIGRTKSISSR 161V/172A, kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK 131M/442I kgtemiwdpngwtgtdnktsik QDIVGINEWSGYSGSFVMHPEL gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN tgldciVpcfwvelirgrpeen TIWTSGSIISFCGVNSDTVGWS tiwtsgsIisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:22) NO:22) VKLAGNSSLCPVSGWAPLSKNN vklagnsslcpvsgwaPLskNn AVRIGSKGDVFVIREPFISCS? Avrigskgdvfvirepfiscsp LECRMFFLTQGALLNDKHSNGT Ni lecrMffltgga11ndkhsnqt IKDRSPYRTLMSVPIGSVPSPY numbering: ikdrspyrtlmsVpigSvpspy 182 Ni- NARFESIAWSASACHDGINWLT 99P/177I/19 nArfesIawsasachdginwlt Mil5 cl IGITGPDSGAVAILKYNGIITD 6T/2 051, igiTgpdsgavaIlkyngiitd 55_D103 TIKSWRNNILRTQESECACVNG 165S, tikswrnni1rtgesecacvng 90-100%
N S105A SCFTIMTDGPSDGQASYKIFRI 100L/408M/4 scftimtdgpsdggasykifri T131M_ EKGKIIKSVEMKAPNYHYEECS 19V, ekgkiiksvemkapnyhyeecs 4421 CYPDSSEITCVCRDNWHGSNR? 161V/172A, cypdsseitcvcrdnwhgsnrp WVSENQNLEYQMGYICSGVFGD 103N/1051\, wysfngnleygmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 131N/442I nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK kgfemiwdpngwtgtdnkfsik SUBSTITUTE SHEET (RULE 26) QDIVGINEWSGYSGSFVMHPFL
gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSIISFCGVNSDTVGWS
tiwtsgsIisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:23) NO:23) VKLAGNSSLCPVSGWAPLAKDN
vkiagnssIcpvsgwaPLAkdn SVRIGSKGDVEVIREPFISCSP
svrigskgdvfvirepfiscsp LECRMFFLTQGALLNDKHSNGT
lecrMffitqgalindkhsngt IKDRSPYRTLMSVPLGSVPSPY
ikdrspyrtimsVpLgSvpspy NARFESIAWSASACHDGINWLT Ni nArfesIawsasachdginwlt IGITGPDSGJAVAILKYNGIITD numbering: igiTgpdsgavaIlkyngiitd 183 N1- TIKSWRNNILRTQESECACVNG 99P/177I/19 tikswrnni1rtqesecacvng Mil5 cl SCFTIMTDGDSDGQASYKIFRI 6T/205I, scftimtdgpsdgqasykifri 55 STO1 EKGKIIKSVEMKAPNYHYEECS 165S, ekgkiiksvemkapnyhyeecs A T131M CYPDSSEITCVCRDNWHGSNR? 100L/408M/4 cypdsseitcvcrdnwhgsnrp 95-100%
I163L WVSENQNLEYQMGYICSGVEGD 19V, wysfnqnleyqmgyicsgvfgd S442I_5 NPRPNDKTGSCGPVSSNGANGV 161V/172A, nprpndktgscgpvssngangv kgfsfrcygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK 631/442I/44 kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL IA
cidivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSIIAFCGVNSDTVGWS
tiwtsgsI1Afcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:24) NO:24) VKLAGNS SLCPVS GWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGEIEVIREPEISCS?
svrigskgEifvirepfiscsp LECRTFELTQGALLNDKHSNGT
lecrtffltqga1lndkhsngt IKDRSPYRTLMSCPIGSPPSPT
ikdrspyrt1msepigSPpspT
NSRFESIAWSASACHDGINWLT
nsrfesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavailkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtqesecacvng 185 N1- SCFTIMTDGPSDGQASYKIFRI 99P/1771/19 scftimtdgpsdgqasykifri Mi175 DF EKGKIIKSVEMKAPNYHYEECS 6T/205I, ekgkiiksvemkapnyhyeecs 2T1 Vii CYPDSSEITCVCRDNWHGSNR? 113E/170T, cypdsseitcvcrdnwhgsnrp RCA
41 V166 WVSENQNLEYQMGYICSCVFCD 165S, wvsfnqnleyqmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 100L/4 08M/4 nprpndktgscgpvssugangv KGFSTKYGNGVWIGRTKSISSR 19V, 453T, kgtsflygngvwigrtksissr kgtemiwdpngwtgtdnktsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpei TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTTGWS
tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pttidk (SEQ ID
NO:25) NO:25) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGEVEVIREPFISCS?
svrigskgEvfvirepfiscsp LECRQFFLTQGALLNDKHSNGT
lecrQff1tqgailndkhsngt IKDRSPYRTLMSCPIGSVPSPT
ikdrspyrtimscpigSvpspT
NSRFESIAWSASACHDGINWLT Ni nsrfesTawsasachdginwlt IGITGPDSGAVAILKYNGIITD numbering: igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG 99P/1771/19 tikswrnniirtgesecacvng 194¨N1- Mill DF SCFTIMIDGPSDGQASYKIFRI 6T/205I, scftimtdgpsdgqasykifri 2TI T,13 EKGKIIKSVEMKAPNYHYEECS 113E/170T, ekgkiiksvemkapnyhyeecs 70-80%
1Q CYPDSSEITCVCRDNWHGSNRP 165S, cypdsseitcvcrdnwhgsnrp WVSENQNLEYQMGYICSGVFGD 100L/4 08M/4 wvsfnqnleyqmgyicsgvfgd NRRPNDKTGSCGPVSSNGANGV 19V, 453T, nprpndktgscgpvssngangv kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHREL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTTGWS
tiwtsgssisfcgvnsdtTgws SUBSTITUTE SHEET (RULE 26) WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:26) NO:26) VKLAGNSSLCPVSGWAPLSKNN vklagnsslcpvsgwaPLskNn AVRIGSKGEVFVIREPFISCS? AvrigskgEvfvirepfiscsp LECRQFFLTQGALLNDKHSNGT lecrQffltggallndkhsngt IKDRSPYRTLMSCPIGSVPSPT ikdrspyrt1mscpigSvpspT
NSRFESIAWSASACHDGINWLT nsrfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD igiTgpdsgavaIlkyngiitd numbering:
TIKSWRNN1LRTQESECACVNG tikswrnni1rtgesecacvng 195 Ni- SOFTIMTDGPSDGQASYKIFRI scttimtdgpsdggasykitri Mi15 DF EKGKIIKSVEMKAPNYHYEECS 6T/2051, ekgkiiksvemkapnyhyeecs 2TI D10 CYPDSSEITCVCRDNWHGSNR? 113E/170T, cypgsseitcvcrdnwhgsnrp 50-60%
3N S105 WVSFNQNLEYQMGYICSGVFGD ibbS' 100L/4 08M/4 wvsfncinleygmgyicsgvfgd A T131Q NPRPNDKTGSCGPVSSNGANGV 19V 103N/105A, 453T nprpndktgscgpvssngangv , KGFSFKYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK T131Q kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN tglggiVpcfwvelirgrpeen T1WTSGSSISFCGVNSDTTGWS tiwtsgssisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:27) NO:27) VKLAGNSSLCPVSGWAPLSKDN vklagnsslcpvsgwaPLskdn SVRIGSKGEIFVIREPFISCS? svrigskgEIfvirepfiscsp LECRQFFLTQCALLNDKHSNGT lecrQffltggallndkhsngt IKDRSPYRTLMSCPIGSPPSPT ikdrspyrt1mscpigSPpspT
NSRFESIAWSASACHDGINWLT Ni nsrfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD igiTgpdsgavaIlkyngiitd numbering:
TIKSWRNNILRTQESECACVNG tikswrnni1rtgesecacvng 196 Ni- SCFTIMTDGPSDGQASYKIFRI scftimtdgpsdggasykifri Mil5 DF EKGKIIKSVEMKAPNYHYEECS 6T/205I, 113E/170T , .. ekgkiiksvemkapnyhyeecs 2TI V11 CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp 40%
41V166 WVSENQNLEYQMGYICSGVFGD 165S, 100L/408M/4 wvsfngnleygmgyicsgvfgd P T131Q NPRPNDKTGSCGPVSSNGANGV 19V 453T nprpndktgscgpvssngangv , , KGFSFKYGNGVWIGRTKSISSR 1141/166P , kgtstkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN tglgciVpctwvelirgrpeen TIWTSGSSISFCGVNSDTTGWS tiwtsgssistcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:28) NO:28) VKLAGNSSLCPVSGWAPLSKDN vklagnsslcpvsgwaPLskdn SVRIGSKGEVFVIREPFISCS? svrigskgEvfvirepfiscsp LECRUF=WALLNDKHSNGT lecrQffltgga11ndkhsngt IKDRSPYRTLMSCPIGSVPSPT ikdrspyrt1mscpigSvpspT
NSRFESIAWSASACHDGINWLT nsrfesIawsasachdginwit IGITGPDSGAVAILKYNGIITD Ni igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtgesecacvng 198 Ni- SCFTIMTDGPSDGQASYKIFRI 99P/177I/19 scftimtdgpsdggasykifri Mi15 DF EKGKI1KSVEMKAPNYHYEECS 61/2051, ekgkiiksvemkapnyhyeecs 2TI T13 CYPDSSEITCVCRDNWHGSNR? 113E/170T, cypdsseitcvcrdnwhgsnrp 20-30%
1Q_5442 WVSFNQNLEYQMGYICSGVFGD 1655, wvsfngnleygmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 100L/4 08M/4 nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR 19V, 453T, kgfsfkygngvwigrtksissr KGFEMINDPNGWTGTDNKFSIK T131Q/442I kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN tgldciVpcfwvelirgrpeen TIWTSGSI1SFCGVNSDTTGWS tiwtsgsIisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NU:29) NU:29) 201 Ni- VKLAGNSSLCPVSGWAPLSKDN Ni vklagnsslcpvsgwaPLskdn Mi15_DF SVRIGSKGEVFVIREPFISCS? numbering: svrigskgEvfvirepfiscsp SUBSTITUTE SHEET (RULE 26) 2T1 T13 LECRMFFITQGALLNDKHSNGT 990/177I/19 lecrMffltqqallndkhsnqt 1M S442 IKDRSPYRTLMSCPIGSVPSPT 6T/2 051, ikdrspyrtlmscpigSvpspT
NSRFESIAWSASACHDGINWLT 113E/170T, nsrfesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD 1653, igiTgpdsgavaIlkyngiitd TIKSWRNNILRTQESECACVNG 100L/4 08M/4 tikswrnnilrtqesecacvng SCETIMTDGPSDGQASYKIFRI 19V, 453T, scftimtdgpsdgclasykirri EKGKIIKSVEMKAPNYHYEECS T131M/442I ekgkiiksvemkapnyhyeecs CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp WVSENQNLEYQMGYICSGVFGD
wvsfnqnleyqmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSIISFCGVNSDTTGWS
tiwtsgsIisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:30) NO:30) VKLAGNSSLCPVSGWAPLSKNN
vklagnsslcpvsgwaPLskEn AVRIGSKGEVEVIREPFISCS?
AvrigskgEvfvirepfiscsp LECRMFFLTQGALLNDKHSNGT
lecrMffltqgallndkhsngt IKDRSPYRTLMSCPIGSVPSPT
ikdrspyrtlmscpigSvpspT
NSRFESIAWSASACHDGINWLT nsrfesIawsasachdginw1t IGITGPDSGAVAILKYNGIITD
igiTgpdsgavaIlkyngiitd numbering:
202 Ni-TIKSWRNNILRTQESECACVNG
tikswrnnilrtqesecacvng Mil5 OF SCETIMIDGPSDGQASYKIFRI
6T/205I, scftimtdgpsdggasykifri 113E/170T, ekgkiiksvemkapnyhyeecs 3N 3105 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 20%
A T131M WVSENQNLEYQMGYICSGVEGD 100L/408M/4 wvsfrIgnieYclmgYiesgvfgd NPRPNDKTGSCGPVSSNGANGV 19V 453T 103N/105A, nprpndktqscgpvssnganqv , KGFSFKYGNGVWIGRTKSISSR
kqfsfkyqnqvwigrtksissr , KGFEMIWDPNGWIGTDNKFSIK T131M/442I kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpel TOLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSIISFCGVNSDTTGWS
tiwtsgsIisfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:31) NO:31) VKLAGNSSLCPVSGWAPLARDN
vklagnssIcpvsgwaPLAkdn SVRIGSKGEVFVIREPFISCSP
svrigskgEvtvireptiscsp LECRMFFITQGALLNDKHSNGT
lecrMffltqgallndkhsngt IKDRSPYRTLMSCPLGSVPSPT
ikdrspyrtlmscpLgSvpspT
NSRFESIAWSASACHDGINWLT Ni nsrfesIawsasachdginwlt IGITGPDSGAVAILKYNGIITD numbering: igiTgpdsgavaIlkyngiitd 203 Ni- TIKSWRNNILRTQESECACVNG 990/1771/19 tikswrnnilrtqesecacvng M115 DF SCFTIMTDGPSDGQASYKIFRI 6T/205I, scftimtdgpsdggasykifri 2TI S10 EKGKIIKSVEMKAPNYHYEECS 113E/170T, ekgkiiksvemkapnyhyeecs 1A T131 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 90%
M I163L WVSENQNLEYQMGYICSGVEGD 100L/408M/4 wvsfnqnleyqmgyicsgvfgd S442I NPRETDKTGSCGEWSSNGANGV i9V, 4530, nprpndktgscgpvssngangv S444A KGFSFKYGNGVWIGRTKSISSR 101A/163L/T kgfsfkygngvwigrtksissr KGFEMIWDPNGWIGTDNKFSIK 131N/442I/4 kgtemiwdpngwtgtdnktsik gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSIIAFCGVNSDTTGWS
tiwtsgsIiAfcgvnsdtTgws WPDGAELPFTIDK (SEQ ID wpdgaeIpttidk (SEQ ID
NO:32) NO:32) eyrnwskpqcnitgfapfskdn SIRLSAGGDIWVTREPYVSCD? sirlsaggdiwvtrepyvscdp 50%
249 N2- WisIT)5 V DKCYQFALGQGTTLNNVHSNDT numbering.dkcyqfalgqgttlnnvhsndt closed VHDRTPYRTLLMNELGQPFHLG
vhdrtpyrtilmne1gQpfh1g (509z:
165Q ( TKQVCIAWSSSSCHDGK mutationsAWLHV
tkqvciawsssschdgkaw1hv open) CVTGDDKNATASFIYNGRLVDS promote cvtgddknatasfiyngrdvds SUBSTITUTE SHEET (RULE 26) IVSWSKEILRTQESECVCINGT open state ivswskeilrtgesecycingt CTVVMTDGSASGKADTKILFIE of NA) ctvv=dgsasgkadtkilfie EGKIVHISTLSGSAQHVEECSC
egkivhtst1sgsaqhveecsc YPRYLGVRCVCRDNWKGSNRPI
yprylgvrcvcrdnwkgsnrpi VDINIKDYSIVSSYVCSGLVGD
vdinikdysivssyvcsgivgd TRRKNDSSSSSHCLDRNNEEGG
tprkndssssshcldpnneegg HGVKGWAFDDGNDVWMGRTISE
hgvkgwafddgndvwmgrtise KLRSGYETFKVIEGWSNPNSKL
klrsgyetfkviegwsnpnskl QINRQVIVDRONRSGYSGIFSV
qinrqvivdrgnrsgysgifsv EGKSCINRCFYVELIRGRKEET
egkscinrcfyvelirgrkeet EVLWTSNSIVVFCGTSGTYGTG
evlwtsnsiyvfcgtsgtygtg SWEDGADINLMPI (SEQ ID swpdgadinimpi (SEQ ID
NO:33) N0:33) EYRNWSKPQCNITGFAPFSKDN
eyrnwskpqcnitgfapfskdn SIRLSAGGDIWVTREPYVSCD?
sirlsaggdiwvtrepyvscdp DKCYQFALGQGTTLNNVHSNDT
dkcyqfalgqgttinnvhsndt VHDRTPYRTLLMNELGQPFHLG
vhdrtpyrtilmnelgQpfhlg TKQVCVAWSSSSCHDGKAWLHV
tkqvcVawsssschdgkawlhv cvSgddknatasfiyngrlvds IVSWSKEILRTQESECVCINGT
ivswskeiirtqesecycingt 255 N2- CTVVMTDGSASGKADTKILFIE numbering:ikn ctvv=dgsasgkadtkilfie 0%
Wis05 V EGKIVHTSTLSGSAQHVEECSC
egkivhtst1sgsaqhveecsc closed yprylgvrcvcrdnwkgsnrpi (100%
76V T19 VDINIKDYSIVSSYVCSGLVGD (mutations vdinikdysivssyvcsglvgd e 53 TPRKNDSSSSSHCLDPNNEEGG promot tprkndssssshcidpnneeqg open) tat HGVKGWAFDDGNDVWMGRTISE opens e hgvkgwafddgndvwmgrtise of NA) KLRSGYETEKVIEGWSNPHSKL
klrsgyetfkviegwsnpnskl QINRQVIVDRGNRSGYSGIFSV
qinrqvivdrgnrsgysgifsv EGKSCINRCFYVELIRGRKEET
egkscinrcfyvelirgrkeet EVLWTSNSIVVFCGTSGTYGTG
evlwtsnsivvfcgtsgtygtg SWPDGADINLMPI (SEQ ID swpdgadinlmpi (SEQ ID
NO:34) N0:34) HFMNNTEALCDAKGFAPFSKDN
hfmnntealcdakgfapfskdn GIRIGSRGHVEVIREPFVSCS?
girigsrghvtvireptvscsp TECRTFFITQGSLLNDKHSNCT
tecrtffltqgsllndkhsngt VHDRSPYRTLMSVEIGSSPNVY
vkdispyrt1msyeigSspnvy QARFEAVAWSATACHDGKKWMT
qarteavawsatachdgkkwmt IGVTGPDAKAVAVVHYGGIPTD
igvtgpdakavavvhyggiptd VINSWAGDILRTQESSCTCIQG
vinswagdilrtqessctcigg 282¨N8-Jian ECFWVMTDGPANRQAQTRAFKA ecfwvmtdgpanrqaqyrafka gxi kqgkivgqaeisfngghieecs Donghu2 CYPNEGKVECVCKDNWTGTNR? numbering: cypnegkvecvckdnwtgtnrp 60%

vlvispdlsyrvgylcaglpsd tprgedsqftgsctspmgnqgy gvkgfgfrqgndvwmgrtisrt SRSGFEILKVRNGWVQNSKEQI
srsgfeilkyrngwvqnskeqi KRQVVVDNLNWSGYSGSFTLPA
krqvvvdninwsgysgsftlpa ELTKRN01,V20FWVEMIRGNPE
eltkrnelvpcfwvemirgnpe EKTIWTSSSSIVMCGVDHEIAD
ektiwtssssivmcgvdheiad WSWHDGAILPFDIDKM (SEQ wswhdgaiiptdidkm (SEQ
ID N0:35) ID K0:35) HFMNNTEALCDAKGFAPFSKDN
hfmnn7ealcdakgfapfskdn GIRIGSRGHVEVIREPFVSCS?
girigsrghvfvirepfvscsp 285¨N8- TECRTFELTQGSLLNDKHSNGT
tecrtffitqgs1indkhsnqt Jiangxi VKDRSPYRTLMSVPIGSSPNVY vkdrspyrtlmsvPigSspnvy QARFEAVAWSATACHDGKKWMT
garfeayawsatachdqkkwmt Donghu2 100% numbering:
013 Elf IGVTGPDAKAVAVVHYGGIPTD
igvtgpdakavavvhyggiptd 160P, 163S
VINSWAGDILRTQESSCTCIQG
yinswagdi1rtgessctciog ecfwvmtdgpanrqaqyrafka Q
KQGKIVGQAEISENGGHIEECS
kqgkivgqaeisfngghieecs CYPNEGKVECVCKDNWTGTNR?
cypnegkvecvckdnwtgtnrp SUBSTITUTE SHEET (RULE 26) VLVISPDISYRVGYLCAGLPSD vlvispdlsyrvgylcaglpsd TPRGEDSQFTGSCTSPMGNQGY tprgedsqftgsctspmgnqgy GVKG7GFRQGNDVWMGRTISRT gvkgfgfrqgndvwmgrtisrt SRSGFEIIEVRNGWVOSKEQI srsgfeilkyrngwygnskeqi KRQVVVDNLNWSGYSGSFTLPA krqvvvdninwsgysgsftipa ELTKRNC=WPCFWVEMIRGNPE eitkrncivpcfwvemirgnpe EKTIWTSSSSIVMCGVDHEIAD ektiw7ssssivmcgvdheiad WSWHDGAILPFDIDKM (SEQ wswhdgailpfdidkm (SEQ
ID NO:36) ID NO:36) HFMNNTEALCDAKGFAPFSKDN htmnntealcdakgtaptskdn GIRIGSRGHVFVIREPFVSCS? girigsrghvfvirepfvscsp TECRTFFITQGSLLNDKHSNGT tecrtff1tqgs1lndkhsngt VKDRSPYRTLMSVPIGSSPNVY vkdrspyrt1msvPigSspnvy QARFEAIAWSATACHDGKKWMT garfealawsatachdgkkwmt IGVTGPDAKAVAVVHYGGIPTD igvtgpdakavavvhyggiptd Jiangxi VINSWAGDILRTQESSCTCIQG vinswagdi1rtgessctciqg ECFWVMIDGPANRQAQYRAFKA ecfwvmtdgpanrciagyrafka KQGKIVGQAEISFNGGHIEECS kqgkivgqaeisfngghieecs Donghu2 numbering:
CYPNEGKVECVCKDNWTGTNRP cypnegkvecvckdnwtgtnrp 013 E16 160P, 163S, VLVISPDLSYRVGYLCAGLPSD v1vispd1syrvgylcaglpsd TPRGEDSQFTGSCTSPMGNQGY tprgedsqftgsctspmgnqgy GVKGFGFRQGNDVWMGRTISRT gvkgtgtrqgndvwmgrtisrt SRSGFEILKVRNGWVQNSKEQI srsgfeilkvrngwvqnskeqi KRQVVVDNLNWSGYSGSFTLPA krqvvvdnlnwsgysgsft1pa ELTKRNCLVPCFWVEMIRGNPE eltkrnclvpdfwvemirgnpe EKTIWTSSSSIVMCGVDHEIAD ektiwt.ssssivmcgvdheiad WSWHDGAILPFDIDKM (SEQ wswhdgailpfdidkm (SEQ
ID NO:37) ID NO:37) HEMNNTEALCDAKGFAPFSKDN hfmnnzealcdakgfapfskdn GIRIGSRGHVFVIREPFVSCS? girigsrghvfvirepfvscsp TECRTFFITQCSLLNDKHSNGT tecrtffitqgs1indkhsngt VHDRSPYRTLMSVPIGSSPNVY vkdrspyrt1msvPigSspnvy QARFEAIAWSATACHDGKKWMT garfealawsatachdgkkwmt 289_N8- IGVTGPDAKAVAIVHYGGIPTD igvtgpdakavaIvhyggiptd Jiangxi VINSWAGDILRTQESSCTCIQG vinswagdi1rtgessctcidg ECFWVMTDGPANRQAQYRAFKA ecfwvmtdgpanrqaqyLafka Donghu2 KQGKIVGQAEISFNGGHIEECS kqgkivgqaeistngghieecs 013 El6 CYPNEGKVECVCKDNWTGTNR? numbering:
cypnegkvecvckdnwtgtnrp /0%
160P, 163S, vivispdisyrvgylcagipsd 1751, 2031 Q165S- TPRGEDSQFTGSCTSPMGNQGY tprgedsqftgsctspmgnqgy V176I- GVKGFGFRQGNDVWMGRTISRT gvkgfgfrqgndvwmgrtisrt srsgteilkvrngwvqnskeqi KRQVVVDNLNWSGYSGSFTLPA krqvvvdnlnwsgysgsft1pa ELTKRNCLVPCFWVEMIRGNPE eltkrnclvpcfwvemirgnpe EKTIWTSSSSIVMCGVDHEIAD ektiwt.ssssivmcgvdheiad WSWHDGAILPFDIDKM (SEQ wswhdgailpfdidkm (SEQ
ID NO:38) ID NO:38) VILTGNSSLCPISGWAPLAKDN viltgnsslcpisgwaPLAkdn SIRIGSKGDVFVIREPFISCSH Sirigskgdvfvirepfiscsh Ni 315 N1- LECRMEFITQGALLNDKHSNGT lecrMff1tqqa11ndkhsngt numbering.
BrevMis VKDRSPYRTLMSVPLGSVPSPY 990/1771/19 vkdrspyrtlmsVpLgSVpspy s1918 c NARFESIAWSASACHDGMGWLT nArfesIawsasachdgmgwlt 155 S10 IGITGPDNGAVAILKYNGIITD 6T/205I, igiTgpdngavaIlkyngiitd 1A G105 TIKSWRNNILRTQESECACVNG 165S tikswrnni1rtgesecacvng 20-30%
S T131M SCFTIMIDGPSNWASYKILKI scftimtdgpsngqasykiiki Vi 63L EKGKVTKSIELNAPNYHYEECS 19V, ekgkv.7Asielnapnyhyeecs Al66V CYPDTGKVMCVCRDNWHGSNRP 161V/172A, cypdtgkvmcvcrdnwhgsnrp 4421 S4 WVSFDQNLDYQIGYICSGVFGD 63L/442I/44 wvsfdqn1dygigyicsgvfgd nprpndgtgscgpvssngangi IA, 105S
KGFSFRYDNGVWIGRTKSTSSR kgfsfrydngvwigrtkstssr SGFEMIWDPNGWTETDSSFSVR sgfemiwdpngwtetdssfsvr SUBSTITUTE SHEET (RULE 26) QDIVAITDWSGYSGSFVMHPEL
gdivaitdwsgysgsfvMhpel TGLDCMVDCFWVELIRGQPKEN
tgldcmVpcfwvelirgqpken TIWTSGSTIAFCGVNSDTVGWS
tiwtsgsIiAfcgvnsdtvgws WPDGAELPFSIDK (SEQ ID wpdgaelpfsidk (SEQ ID
NO:39) NO:39) VKLAGNSSLCPINGWAPLSKDN
vklagnssIcpingwaPLskdn SVRIGSKGDVFVIREPFISCSH
sVrigskgdvfvirepfiscsh LECRQFFLTQGALLNDKHSNGT lecrQffltwa1lndkhsngt VKDRSPHRTLMSVPIGSVPSPY
vkdrsphrt1msVpIgSVpspy NARFESIAWSASACHDGTSWLT nArtesIawsasachdgtsw1t IGITGPDNGAVAILKYNGIITD
igiTgpdngavaIlkyngiitd numbering:
TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng 354 Ni- 992/1171/19 SCFTVMIDGPSNGQASYKIFKM
scftvmtdgpsngqasykifkm VN04 cl 61/205I, EKGKVVKSVELDAPNYHYEECS
ekgkvvksveldapnyhyeecs 55 1106 1653, CYPNAGEITCVCRDNWHGSNR?
cypnageitcvcrdnwhgsnrp 100%

WVSENQNLEYQIGYICSGVEGD wvsfnqnleygigyicsgvfgd 19V, V163I¨ NPRPNDGTGSCGPVSSNGAYGV
nprpndqtqscgpvssnqayqv A166V 161V/172A, KGESFKYGNGVWIGRTKSTNSR
kgfsfkygngvwigrtkstnsr 131Q, 106V, SGFEMIWDPNGWTETDSSFSVK
sgfemiwdpngwtetdssfsvk 1631, 166V
QDIVAITDWSGYSGSFVMHPEL
gdivaitdwsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKES
tgldciVpcfwvelirgrpkes TIWTSGSSISFCGVNSDTVGWS
tiwtsgssistcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
N :40) NO:40) VKLAGNSSLCPINGWAPLSKDN
vklagnsslcpingwaPLskdn SVRIGSKGDIFVIREPFISCSH
sVrigskqdIfvirepfiscsh LECRQFELTQGALLNDKHSNGT
lecrQffltqgallndkhsngt VKDRSPHRTLMSVPIGSPPSPY
vkdrsphrt1msVpIgSPpspy NARFESIAWSASACHDGTSWLT Ni nArfesIawsasachdgtswit IGITGPDNGAVAILKYNGIITD numbering: igiTgpdngavaIlkyngiitd 356 N1- TIKSWRNNILRTQESECACVNG 99P/1771/19 tikswrnni1rtqesecacvng VN04 cl SCFTVMIDGPSNGQASYKIFKN 6T/2051, scftvmtdgpsngqasykifkm 55 I106 EKGKVVKSVELDAPNYHYEECS 1653, ekgkvvksveldapnyhyeecs V_V111I CYPNAGEITCVCRDNWHGSNR? 100L/408M/4 cypnageitcvcrdnwhgsnrp BO%
T131Q WVSENQNLEYQIGYICSCVECD 19V, wvsfnqnleygigyicsgvfgd V163I_V NPRPNDGTGSCGPVSSNGAYGV 161V/1721%, nprpndgtgscgpvssngaygv 166P KGFSTKYGNGVWIGRTKSTNSP 131Q, 106V, kgtstkygngvwigrtkstnsr SGFEMIWDPNGWTETDSSFSVK 1631, sgtemiwdpngwtetdsstsvk gdivaitdwsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKES
tgldciVpcfwvelirgrpkes TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:41) NO:41) VKLAGNSSLCPINGWAPLSKDN
vklagnsslcpingwaPLskdn SVRIGSKGDIFVIREPFISCS? sVrigskgdIfvirepfiscsP
Ni LECRQFFLTQGALLNDKHSNGT
lecrQff1tqga11ndkhsngt numbering:
VKDRSPHRTLMSVPIGSPPSPY 99P/1771/19 vkdrsphrt1msVpIgSPpspy nArfesIawsasachdgtswlt 61/2051, VN04 cl IGITGPDNGAVAILKYNGIITD
igiTgpdngavaIlkyngiitd 165S, tikswrnni1rtgesecacvng scftvmtdgpsnggasykifkI
19V, ,, 70%

ekgkIvksveMdapnyhyeecs 161V/1,2A, V1631 V CYPNAGEITCVCRDNWHGSNR?
cypnageitcvcrdnwhgsnrp 131Q, 106V, 166P sm WVSENQNLEYQIGYICSGVEGD
wvsfnqnleygigyicsqvfqd 1631, al1Ca10 NPRPNDGTGSCGPVSSNGAYGV 1141/166D, nprpndgtgscgpvssngaygv 9pack KGFS7KYGNGVWTGRTKSTNSP
kgfsfkygngvwigrtkstnsr 126?, 210G, SGFEMIWDPNGWTETDSSFSVK
sgfemiwdpngwtetdssfsvk 2571, 2621, QDIVAITDWSGYSGSFVMHPEL gdivaitdwsgysgsfvMhpe1 TGLDCIVDCFWVELIRGRPKES
tgldciVpcfwvelirgrpkes TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws SUBSTITUTE SHEET (RULE 26) WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:42) NO:42) VILTGNSSLCPIRGWAPLSKDN viltgnsslopirgwaPLskdn SVRIGSKGDVFVIREPFISCSH SVrigskgdvfvirepfiscsh LECRTFFITQGALLNDKHSRGT lecrtffltqga11ndkhsrgt FKDRSPYRTLMSVPIGSVPSPY fkdrspyrTimsVpIgSVpspy NARFESIAWSASACHDGMGWLT Ni nArfesIawsasachdgmgw1t IGITGPDDGAVAILKYNGIITE numbering: igiTgpddgavaIlkynGiite TIKSWRKNILRTQESECTCVNG 99P/1771/19 tikswrkni1rtqesectcvng WSN33 c SOFTIMTDGPSDGLASYKIFKI 6T/2 051, scttimtdgpsdglasykitki EKGKVTKSIELNAPNSHYEECS 165S, ekgkvtksielnapnshyeecs CYPDTGKVMCVCRDNWHGSNR? 100L/408M/4 cypdtgkvmcvcrdnwhgsnrp BO%

WVSFDQNIDYKIGYICSGVFGD 19V, wvsfdqn1dykigyicsgvfgd NPRPKDGTGSCGPVSADGANGV 161V/172A, nprpkdgtgscgpvsadgangv A166V-R KGFSYKYGNGVWIGRTKSDSSR 105S, 106V, kdfsykydngvwigrtksdssr HGFEMIWDPNGWTETDSRFSMR 1571, 1631, hgfemiwdpngwtetdsrfsmr QDVVAITNRSGYSGSFVMHPEL 166V, 210G gdvvaitnrsgysgsfvMhpel TGLDCMVPCFWVELIRGLPEED tgldcmVpcfwvelirglpeed AIWTSGS1ISFCGVNSDTVDWS aiwtsgsiisfcgvnsdtvdws WPDGAELPFTIDK (SEQ ID wpdgaeipftidk (SEQ ID
NO:43) NO:43) VILTGNSSLCPIRGWAPLSKDN viltgnsslcpirgwaPLskdn SVRIGSKGDVFVIREPFISCSH SVrigskgdvfvirepfiscsh LECRQFFLTQGALLNDKHSRGT lecrQffltqgallndkhsrgt FKDRSPYRTLMSVPIGSVPSPY Ni fkdrspyrT1msVpIgSVpspy NARFESIAWSASACHDGMGWLT nArfesIawsasachdgmgw1t numbeLing:
367 N1- IGITGPDDGAVAILKYNGIITE igiTgpddgavaIlkynGiite WSN33 c TIKSWRKNILRTQESECTCVNG tikswrkni1rtqesectcvng 6T/205I, 155 G10 SOFTIMTDGPSDGLASYKIFKI scftimtdgpsdglasykifki 5S 1106 EKGKVTKSIELNAPNSHYEECS 165S, ekgkvtksielnapnshyeecs V 1131Q CYPDTGKVMCVCRDNWHGSNR? cypdtgkvmcvcrdnwhgsnrp A1571 WVSFDQNIDYKIGYICSGVFGD 19V, wvsfdqn1dykigyicsgvfgd 161V/172A, T/163I A NPRPKDGTGSCGPVSADGANGV nprpkdgtgscgpvsadgangv 131Q, 105S, 166V R2 KGFSYKYGNGVWIGRTKSDSSR kgtsykygngvwigrtksdssr 106V, 157T, 10G HGFEMIWDPNGWTETDSRFSMR hgfemiwdpngwtetdsrfsmr 1631, 166V, QDVVAITNRSGYSGSFVMHPEL qdvvaitntsgysgsfvMhpe1 TGLDCMVPCFWVELIRGLPEED tgldcmVpctwvelirglpeed AIWTSGSIISFCGVNSDTVDWS aiwtsgsiistcgvnsdtvdws WPDGAELPFTIDK (SEQ ID wpdgaeipftidk (SEQ ID
NO:44) NO:44) VILTGNSSLCPIRGWAPLSKDN viltgnsslcpirgwaPLskdn SVRIGSKGDVFVIREPFISCSH SVrigskgdvfvirepfiscsh LECRQFFIXQGALLNDKHSRGT iecrQff1tqga11ndkhsrgt FKDRSPYRTLMSVPIGSVPSPY fkdrspyrT1msVpIgSVpspy Ni NARFESIAWSASACHDGMGWLT nArfesIawsasachdgmgw1t 369 N1- numbering:
IGITGPDDGAVAILKYNGIITE WSN33 c 99P/17-'I/19 igiTgpddgavaIlkynGiite TIKSWRKNILRTQESECTCVNG tikswrkni1rtqesectcvng 155 G10 6T/2051, SCFTIMTDGPSDGLASYKIFKI scftimtdgpsdglasykifki SS 1106 165S, EKGKVTKSIELNAPNSHYEECS ekgkvtksielnapnshyeecs CYPDTGKVMCVCRDNWHGSNR? cypdtgkvmcvcrdnwhgsnrp 70%
A157T 19V, WVSFDQN=DYKIGYICSGVFGD wvsfdqnldykigyicsgvfgd V1631 A 161V/1-1')A, NPRPKDGTGSCGPVSADGANGV nprpkdgtgscgpvsadgangv 166V R2 131Q, 105S, 10G '44 KGFSYKYGNGVWIGRTKSDSSR kgfsykygngvwigrtksdssr 106V, 157T, HGFEMIWDDNGWTETDSRFSMR hgfemiwdpngwtetdsrfsmr 2S 1631, 166V, QDVVAITNRSGYSGSFVMHPEL qdvvaitnrsgysgsfvMhpe1 210G, 442S
TGLDCMVPCFWVELIRGLPEED tgldcmVpcfwvelirglpeed AIWTSGSSISFCGVNSDTVDWS aiwtsgsSisfcgvnsdtvdws WPDGAELPFTIDK (SEQ ID wpdgaeipftidk (SEQ ID
NU:45) NU:45) 371 N1- VILTGNSSLCPIRGWAPLSKDN Ni viltgnssicpirgwaPLskdn WSN33 _c SVRIGSKGDIFVIREPFISCSH numbering: SVrigskgdIfvirepfiscsh SUBSTITUTE SHEET (RULE 26) 155610 LECRQFFLTQGALLNDKHSRCT 99P/177I/19 lecrQffltqqa1lndkhsrqt 5S1106 FKDRSPYRTLMSVPIGSPPSPY 6T/2 051, fkdrspyrT1msVpIgSPpspy V V114I NARFESIAW5ASACHDGMGWLT 1655, nArfesIawsasachdgmgwlt igiTgpddgavaIlkynGiite A1571 V TIKSWRKNILRTQESECTCVNG 19V, tikswrkni1rtgesectcvng 1631 Al SCFTIMTDGPSDGLASYKIFKI 161V/172A, scftimtdgpsdglasykifki 66P R21 EKGKVTKSIELNAPNSHYEECS 1141/166P, ekgkvtksielnapnshyeecs 0G1442 CYPDTGKVMCVCRDNWHGSNR? 1310, 1053, cypdtgkvmcvcrdnwhgsnrp WVSFDQN1DYKIGYICSCVFCD 106V, 157T, wvsfdqn1dykigyicsgvfqd NPRPKDGTGSCGPVSADGANGV 1631, 210G, nprpkdgtgscgpvsadgangv kgfsykygngvwigrtksdssr HGFEMIWDPNGWTETDSRFSMR
hgfemiwdpngwtetdsrfsmr QDVVAITNRSCYSCSFVMHPEL
qdvvaitnrsgysgsfvMhpe1 TCLDCMVPCFWVELIRGLPEED
tgidcmVpcfwvelirglpeed AIWTSGSSISFCGVNSDTVDWS
aiwtsgsSisfcgvnsdtvdws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:46) NO:46) VKLAGNSSLCPVSGWAPLSKDN
vklagnssicpvsgwaPLskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFELTQGALLNDKHSNGT
lecrtff1tqga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAMAVLKYNGIITD
igiTgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG Ni tikswrnni1rtgesecacvng SCFTVMTDGPSNGQASYKIFRI numbering: scftvmtdgpsnggasykifri Cal139- EKGKIVKSVEMNAPNYHYEECS 99P/177I/19 ekgkivksvemnapnyhyeecs 155 12 CYPDSSEITCVCRDNWHGSNR? 61, 1655, cypdsseitcvcrdnwhgsnrp c wvsfncinleygigyicsgifgd NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktqscgpvssncjanqv kgfsfkygngvwigrtksissr NCFEMIWDPNGWIGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpe1 TOLDCIVPCFWVELIRGRPKEN
tqldciVpcfwvelircirpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:47) NO:47) VKLAGNSSLCPVSGWAPLSRDN
vklagnss1cpvsgwaPLskdn SVRIGSKGDVFVIREPFISCSP
svrigskgdvtvireptiscsp LECRTFELTQCALLNDKHSNCT
lecrtffitqqa1indkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESVAWSASACHDGINWLT
nArfesvawsasachdginwit ICITCPDNCAVAVLKYNGIITD
igiTgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG Ni tikswrnni1rtgesecacvng 400 N1- SCFTVMTDGPSNGQASYKIFRI numbering:
scftvmtdgpsnggasykifri Ca109- EKGKIVKSVEMNAPNYHYEECS 99P/196T, ekgkivksvemnapnyhyeecs c15511 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp 77V 120 WVSENQNLEYQIGYICSGIFGD 100L/4 08M/4 wvsfnqnleygigyicsgifgd 5V NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv kgfsfkygngvwigrtksissr NCFEMIWDPNGWTGTDNNESIK
ngtemiwdpngwtgtdnntsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpctwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaeipftidk (SEQ ID
NO:48) NO:48) 401 511- VKLAGN5SLCPV5GWAILSKDN Ni vklagnsslcpvsgwaiLskdn Cai09- SVRIGSKGDVFVIREPFISCS? numbering: svrigskgdvfvirepfiscsp c155 P9 LECRTFFLTQGALLNDKHSNGT 1655, lecrtifitqqa11ndkhsngt 91 1177 IKDRSPYRTLMSVPIGSVPSPY 100L/4 08M/4 ikdrspyrt1msVpigSvpspy V 11955 NARFESVAWSASACHDGINWLT 19V, nArfesvawsasachdginwlt 1205V IGISCPDNGAM7sYLKYNGIITD 161V/172A
igisgpdngavavlkyngiitd SUBSTITUTE SHEET (RULE 26) TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsnggasykifri EKGKIVKSVEMNAPNYHYEECS
ekgkivksvemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp WVSFNQNIEYQIGYICSGIFGD
wvsfncinleygigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSFKYGNGVWIGRIKSISSR
kgfsfkygngvwigrtksissr NGFEMIWDPNGWIGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSOYSGSFVMHPEL
gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:49) NO:49) VKLAGNSSLCPVSGWAIYSKDN
vklagnsslcpvsgwaiyskdn SVRIGSKGDVEVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFITQGALLNDKHSNGT
lecrtffltgga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrtlmsVpigSvpspy NARFESVAWSASACHDGINWLT
nArfesvawsasachdginwit IGISGPDNGAVAVLKYNGIITD
igisgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng Ca109-SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsnggasykifri c155 P9 Ni EKGKIVKSVEMNAPNYHYEECS
ekgkivksvemnapnyhyeecs 91 1177 numbering:
CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp V 1195S 165S, WVSENQN=QIGYICSGIFGD
wvstngnleygigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv no-KGESYKYGNGVWIGRTKSISSR
kgfsfkygngvwigrtksissr spaceB
NGFEMIWDPNGWIGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvghpe1 TCLDCIRPCFWVELIRGRPKEN
tgldcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (3E0 ID wpdgae1pftidk (SEQ ID
NO:50) NO:50) VKLAGNSSLCPVSGWAIYSKDN
vklagnsslcpvsgwaiyskdn SVRIGSKGDIFVIREPFISCSP
svrigskgdItvireptiscsp LECRTFFITQGALLNDKHSNCT
lecrtffitgga1indkhsngt IKDRSPYRTLMSVPIGSPPSPY
ikdrspyrt1msVpigSPpspy NARFESVAWSASACHDGINWLT
nArtesvawsasachdginwIt igisgpdngavavlkyngiitd Ca109-TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng c155 P9 91 1177 SCFTVMTDGPSNGQASYKIFRI Ni scftvmtdgpsnggasykifri EKGKIVKSVEMNAPNYHYEECS numbering: ekgkivksvemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp I205V- WVSENQN=QIGYICSGIFGD 161V/172A, wvsfngnleygigyicsgifgd no-nprpndktgscgpvssngangv spaceB- KGFSFKYGNGVWIGRIKSISSR
kgfsfkygngvwigrtksissr ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvghpe1 TGLDCIRPCFWVELIRGRPHEN
tg1dcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pttidk (SEQ ID
NO:51) NO:51) VKLAGNSSLCPVSGWAIYSKDN
vklagnsslcpvsgwaiyskdn SVRIGSKGDVEVIREPFISCS?
svrigskgdvfvirepfiscsp Ca109-LECRQFFITQGALLNDKHSNGT
lecrQffltgga11ndkhsngt c155 P9 Ni IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy 91J177 numbering:
NARFESVAWSASACHDGINWLT
nArfesvawsasachdginwlt V T195S 165S, IGISGPDNGAMAVLKYNGIITD 1205V 161V/1-'2A, igisgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng no- 131Q
SCFTVMTDUPSNGQASYKIFRI
scftvmtdgpsnggasykifri spaceB- EKGKIVKSVEMNAPNYHYEECS
ekgkivksvemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp SUBSTITUTE SHEET (RULE 26) WVSENQNLEYQIGYICSGIFGD wvsfngnleygigyicsgifqd NPRRNDKTGSCGRVSSNGANGV nprpndktgscgpvssngangv KGFS7KYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL gdivginewsgysgsfvqnpe1 TGLDCIRPCFWVELIRGRPKEN tgldcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:52) NO:52) VKLAGNS SLCPVS GWAIYSKDN vklagnsslcpvsgwaiyskdn SVRIGSKGDIFVIREPFISCS? svrigskgdIfvirepfiscsp LECRQFFLTQGALLNDKHSNGT lecrQffltggallndkhsngt IKDRSPYRTLMSVPIGSDPSPY ikdrspyrt1msVpigSDpspy 405 N1- NARFESVAWSASACHDGINWLT nArfesvawsasachdginwit Ca109- IGISGPDNGAMAYLKYNGIITD igisgpdngavavlkyngiitd c155 P9 TIKSWRNNILRTQESECACVNG N1 tikswrnni1rtgesecacvng 91 1177 SCFTVMTDGPSNGQASYKIFRI scftvmtdgpsngclasykifri / T195S EKGKIVKSVEMNAPNYHYEECS numbering:
ekgkivksvemnapnyhyeecs 165S, 1205V CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp no- WVSENQNLEYQIGYICSGIFGD 1141/166P, wvsfnnleygigyicsgifgd 131Q , ci spaceB NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv V1141 T KGFSFKYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr 131Q V1 NGFEMIWDPNGWTGTDNNFSIK ngfemiwdpngwtgtdnnfsik gdivginewsgysgsfvqhpel TGLDCIRPCFWVELIRGRPKEN tgldcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:53) NO:531 VKLAGNSSLCPVSGWAILAKDN vklagnsslcpvsgwaiLAkdn SVRIGSKGDVFVIREPFISCS? svrigskgdvfvirepfiscsp LECRMFFLTQCALLNDKHSNGT 1eorMff1tqga11ndkhsngt 406 N1- IKDRSFYRTLMSVPLGSVPSPY ikdrspyrt1msVpLgSvpspy Cal 09- NARFESVAWSASACHDGINWLT
nArfesvawsasachdginwit c15.5 P9 IGISGPDNGAMAVLKYNGIITD igisgpdngavavlkyngiitd 91 1177 TIKSWRNNILRTQESECACVNG Ni tikswrnni1rtgesecacvng SCFTVMPDGPSNGQASYKIFRI numbering: scftvmtdgp6ngulasykifti I205V EKGKIVKSVEMNAPNYHYEECS 1655, ekgkivksvemnapnyhyeecs no- CYPDSSEITCVCRDNWHGSNR? 161V/112A, cypdsseitcvcrdnwhgsnrp spaceB WVSENQNLEYQIGYICSGIFGD 100L/1017\/1 wvsfnqnleycligyicsgifqd YlOOL S NPRPNDKTGSCGPVSSNGANGV 3714/163L/44 nprpndktgscgpvssngangv 101A 11 KGFSEKYGNGVWIGRTKSISSR 21/444A kgfsfkygngvwigrtksissr 31M 116 NGFEMIWDPNGWTGTDNNESIK ngfemiwdpngwtgtdnnfsik 3L S442 QDIVGINEWSGYSGSFVQHDEL gdivginewsgysgsfvqhpel I_S444A TGLDCIRPCFWVELIRGRPKEN tgldcirpcfwvelirgrpken TIWTSGSIIAFCGVNSDTVGWS tiwtsgsIiAfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:54) NO:54) VKLAGNSSLCPVSGWAILAKDN vklagnsslcpvsgwaiLAkdn CaltT)9- SVRIGSKGDIFVIREPFISCS? svrigskgdIfvirepfiscsp c155 99 LECRMFFLTQGALLNDKHSNGT lecrMff1tqcia11ndkhsngt 91 1177 IKDRSPYRTLMSVPLGSPPSPY Ni ikdrspyrt1msVpLgSPpspy / 11953 NARFESVAWSASACHDGINWLT numbering: nArfesvawsasachdginwlt -IGISGPDNGAMAVLKYNGIITD 165S, igisgpdngavavlkyngiitd 1205V¨ TIKSWRNNILRTQESECACVNG 161V/172A, tikswrnni1rtgesecacvng no SCFTVMTDGPSNWASYKIFRI 114I/1663, scftvmtdgpsngclasykifri spaceB YlOOL EKGKIVKSVEMNAPNYHYEECS 100L/101A/1 ekgkivksvemnapnyhyeecs S
101A V1 CYPDSSEITCVCRDNWHGSNR? 31M/163L/44 cypdsseitcvcrdnwhgsnrp 141 T13 WVSFNQNLEYQIGYICSGIFGD 21/444A wvsfnqnleygigyicsgifgd 1M 7163 NPRPNDKTUSCGEWSSNGANGV nprpndktgscgpvssngangv L
KGFSFKYGNGVWIGRTKSISSR kgfsf-kygngvwigrtksissr NGFEMIWDPNGWTGTDNNESIK ngfemiwdpngwtgtdnnfsik SUBSTITUTE SHEET (RULE 26) 54421 QDIVGINEWSGYSGSFVQHPEL qdivginewsgysgsfvqhpel tgldcirpcfwvelirgrpken TIWTSGSITAFCGVNSDTVGWS
tiwtsgsTiAfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:55) NO:55) VKLAGNSSLCPVSGWAPLSKDN
vklagnssicpvsgwaPLskdn SVRIGSKGDIFVIREPFISCS?
svrigskgdIfvirepfiscsp LECRTFELTQGALLNDKHSNGT
lecrtffltqgallndkhsngt IKDRSPYRTLMSVPIGSPPSPY
ikdrspyrt1msVpigSPpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD Ni igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtqesecacvng 408 Ni- SCFTVMIDGPSNGQASYKIFRI 99P/1771/19 scftvmtdgpsngclasykifri Ca109- EKGKIVKSVEMNAPNYHYEECS 6T/2 051, ekgkivksvemnapnyhyeecs c155 V1 CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseifcvcrdnwhgsnrp 141 V16 WVSENQNLEYQIGYICSGIFGD 100L/408M/4 wvsfnqnleyqigyicsgifgd 62 NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv KGFSFKYGNGVWIGRIKSISSR 161V/172A, kgfsfkygngvwigrtksissr ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:56) NO:56) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRQFFLTQGALLNDKHSNGT
lecrQffltqga1lndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD Ni igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG numbering: tikswrnni1rtqesecacvng 409 Ni-SCFTVMIDGPSNGQASYKIFRI 992/1771/19 scftvmtdgpsngqasykifri CalT)9- EKGKIVKSVEMNAPNYHYEECS 6T/2051, ekgkivksvemnapnyhyeecs c155 Ti CYPDSSEITCVCRDNWHGSNR? 165S, cypdsseitcvcrdnwhgsnrp wvsfriqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssugangv KGFSTKYGNGVWIGRIKSISSR 161V/172A, kgtsflygngvwigrtksissr ngtemiwdpngwtgtdnntsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:57) NO:57) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGDIFVIREPFISCS?
svrigskgdIfvirepfiscsp LECRQFFLTQGALLNDKHSNGT
lecrQff1tqga11ndkhsngt IKDRSPYRTLMSVPIGSPPSPY ikdrspyrt1msVpigSPpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwlt numbering:
410 Ni-IGITGPDNGAVAILKYNGIITD
igiTgpdngavaIlkyngiitd Ca109- TIKSWRNNILRTQESECACVNG
6T/2051, tikswrnni1rtqesecacvng c155 165S
SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsngqasykifri V1 , 100L/408M/1 ekgkivksvemnapnyhyeecs 1Q V166 CYPDSSEITCVCRDNWHGSNR? 19V, cypdsseitcvcrdnwhgsnrp wvsfnqnleygigyicsgifgd , nprpndktgscgpvssngangv 131Q , KGFS7KYGNGVWIGRIKSISSR kgfsfkygngvwigrtksissr NGFEMIWDPNGWIGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
cidivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws SUBSTITUTE SHEET (RULE 26) WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:59) NO:58) VKLAGNSSLCPVSGWAPLAKDN
vklagnsslcpvsgwaPLAkcin SVRIGSKGDVFVIREPFISCS? svrigskgdvfvirepfiscsp LECRMFFTQGALLNDKHSNGT lecrMffltqga11ndkhsngt IKDRSPYRTLMSVPLGSVPSPY ikdrspyrtimsVpLgSvpspy NARFESIAWSASACHDGINWLT N1 nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD numbering: igiTgpdngavaIlkyngiitd 411 N1- TIKSWRNNILRTQESECACVNG 99P/1771/19 tikswrnni1rtgesecacvng Ca109- SOFTVMIDGPSNGQASYKIFRI 6T/2051, scftvmtdgpsngclasykitri c155 S1 EKGKIVKSVEMNAPNYHYEECS 165S, ekgkivksvemnapnyhyeecs 01A T13 CYPDSSEITCVCRDNWHGSNR? 100L/4 08M/4 cypdsseitcvcrdnwhgsnrp 1M1163 WVSFNQNLEYQIGYICSGIFGD 19V, wvsfnqnleycligyicsgifgd L ,L1421 NPRPNDKTGSCGPVSSNGANGV 161V/172A, nprpndktgscgpvssngangv 5444A KGESTKYGNGVWIGRTKSISSR 101A/131M/1 kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK 63L/442I/44 ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL 4A gdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKEN tgldciVpcfwvelirgrpken TIWTSGSIIAFCGVNSDTVGWS tiwtsgsI1Afcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:59) NO:59) VKLAGNSSLCPVSGWAPLAKDN vklagnsslcpvsgwaPLAkdn SVRIGSKGDIFVIREPFISCS? svrigskgdIfvirepfiscsp LECRMFFLTQGALLNDKHSNGT lecrMffltqga1lndkhsngt IKDRSPYRTLMSVPLGSPPSPY ikdrspyrt1msVpLgSPpspy NARFESIAWSASACHDGINWLT nArfesIawsasachdginwit numbeLing;
412 N1- IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd Ca109- TIKSWRNNILRTQESECACVNG 99P/1771/19 6T/2051 tikswrnni1rtqesecacvng c155 S1 SOFTVMTDGPSNGQASYKIFRI , scftymtdgpsngciasykifri 01A V11 EKGKIVKSVEMNAPNYHYEECS 165S, 100L/4 08M/4 ekgkivksvemnapnyhyeecs 41T131 CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp M I163L WVSFNQNLEYQIGYICSGIFGD 19V, wvsfnqnleycligyicsgifgd \I-166P NPRPNDKTGSCGPVSSNGANGV 161V/172A, nprpndktgscgpvssngangv S4421 S KGFSFKYGNGVWIGRTKSISSR 114I/166P, 101A/131M/1 kgtstkygngvwigrtksissr ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL qdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPREN tgldciVpdtwvelirgrpken TIWTSGSIIAFCGVNSDTVGWS tiwtsgsIiAtcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:60) NO:60) VKLAGNSSLCPVSGWAIYSKDN vklagnsslcpvsgwaiyskdn SVRICSKCDVEVIREPFISCS? svrigskgdvfvirepfiscsp LECRTFELTWALLNDKHSNGT lecrtffltqga11ndkhsngt IKDRSPYRTLMSCPIGSVPSPY ikdrspyrt1mscpigSvpspy NSRFESVAWSASACHDGINWLT nsrfesvawsasachdginwit IGISGPDNGAVAYLKYNGIITD igisgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG tikswrnni1rtqesecacvng SOFTVMTDGPSNGQASYKIFRI scftvmtdgpsngqasykifri Ni- EKGKIVKSVEMNAPNYHYEECS N1 ekgkivksvemnapnyhyeecs Cai09 E CYPDSSEITCVCRDNWHGSNR? numbering: cypdsseitcvcrdnwhqsnrp wvsfnqnleyeligyicsgifgd NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv KGFSYKYGNGVWIGRTKSISSR kgfsYkygngvwfgrtksissr NGFEMIWDDNGWTCTDNNESIK ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQMPEL gdivginewsgysgsfvghpe1 TGLDCIRPCFWVELIRGRPKEN tgldcirperwvelirgrpken TIWTSGSSISFCGVNSDTVGWS tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NU:61) NU:61) SUBSTITUTE SHEET (RULE 26) VKLAGNSSLCPVSGWAIYSKDN
yklagnsslcpvsgwaiyskcin svrigskgdvfvirepfiscsp LECRTFFETQGALLNDKHSNGT
lecrtffltqga1lndkhsngt IKDRSPYRTLMSCPIGTVPSPY
ikdrspyrtimscpigTvpspy NSRFESVAWSASACHDGINWLT
nsrfesvawsasachdginwit IGISGPDNGAVAVLKYNGIITD
igisgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnniirtgesecacvng SOFTVMTDGPSNGOASYKIFRI
scftvmtdgpsnggasykifri Ni- EKGKIVKSVEMNAPNYHYEECS Ni ekgkivksvemnapnyhyeecs Ca109 E CYPDSSEITCVCRDNWHGSNR2 numbering:
cypdsseitcvcrdnwhgsnrp wvsfnqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KCFSYKYGNGVWIGRTKSISSR
kgfsYkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
qdivginewsgysgsfvqhpel TGLDCIRPCFWVELIRGRPKEN
tgldcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pfridk (SEQ ID
NO:62) NO:62) VKLAGNSSLCPVSGWAIYSKDN
yklagnsslcpvsgwaiyskcin SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFETQGALLNDKHSNGT
lecrtttitqgailndkhsngt IKDRSPYRTLMSCPIGVVPSPY
ikdrspyrtimscpigVvpspy NSRFESVAWSASACHDGINWLT
nsrfesvawsasachdginwlt IGISGPDNGAMAYLKYNGIITD
igisgpdngavavlkyngiitd TIKSWRNNIERTQESECAOVNG
tikswrnni1rtgesecacvng SCFTVMTDGPSNGQASYKIFRI
scftumtdgpsngclasykifri Ni- EKGKIVKSVEMNAPNYHYEECS Ni ekgkivksvemnapnyhyeecs Ca109_E CYPDSSEITCVCRDNWHGSNR? numbering: cypdsseitcvcrdnwhgsnrp wvsfnqnleygigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSYKYGNGVWIGRTKSISSR
kgfsYkygngvwigrtksissr NOFEMIWDPNGWTGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvqhpe1 TGLDCIRPCFWVELIRGRPKEN
tgldcirpcfwvelirgrpkem TIWTSGSSISECGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:63) NO:63) VKLAGNSSLCPVSGWAIYSKDN
yklagnsslcpvsgwaiyskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFETQGALLNDKHSNGT
lecrtffitggabindkhsngt IKDRSPYRTLMSCPIGAVPSPY
ikdrspyrtimscpigAvpspy NSRFESVAWSASACHDGINWLT
nsrfesvawsasachdginwlt IGISGPDNGAMAVLKYNGIITD
igisgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnniirtgesecacvng SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsngclasykifri Ni- EKGKIVKSVEMNAPNYHYEECS Ni ekgkivksvemnapnyhyeecs Ca109 E CYPDSSEITCVCRDNWHGSNR2 numbering:
cypdsseitcvcrdnwhgsnrp wvsfnqnleygigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KGFSYKYGNGVWIGRTKSISSR
kgfsYkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdrinfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvqhpe1 TCLDCIRPCFWVELIRGRPKEN
tgldcirpctwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:64) NO:64) Ni-VKLAGNSSLCPVSGWAIYSKDN Ni yklagnsslcpvsgwaiyskdn svrigskgdvfvirepfiscsp Ca109-E LEGRTFF numbering:ETQGALLNDKHSNGT 1651 lecrtffltqga1lndkhsngt IKDRSPYRTLMSCPIGIVPSPY
ikdrspyrtimscpigIvpspy SUBSTITUTE SHEET (RULE 26) NSRFESVAWSASACHDGINWLT
nsrfesvawsasachdginwlt IGISGPDNGAVAVLKYNGIITD
igisgpdngavavlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnni1rtqesecacvng SOFTVMTDGPSNGQASYKIFRI
scftvmtdgpsnomasykifri EKGKIVKSVEMNAPNYHYEECS
ekgkivksvemnapnyhyeecs cypdsseitcvcrdnwhgsnrp WVSENQNLFYQIGYICSGIFGD
wvstrignleygigyicsgitgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KOFSYKYGNOVWIGRTKSISSR
kgfsYkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINFWSGYSGSFVQHPFL
qdivginewsgysgsfvqhpel TGLDCIRPCFWVELIRGRPKEN
tgidcirpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:65) NO:65) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGDVFVIREPFISCSP
svrigskgdvfvirepfiscsp LECRTFELTQGALLNDKHSNGT
lecrtffltqga11ndkhsngt IKDRSPYRTLMSVPIGTVPSPY
ikdrspyrt1msVpigTvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnniirtqesecacvng numbering:
Ni-SCFTVMTDGPSNGQASYKIFRI
scftvmtdgpsngqasykitri Ca109-ekgkivksvemnapnyhyeecs , c155 CYPDSSEITCVCRDNWHGSNR? 165T
cypdsseitcvcrdnwhgsnrp S1 , LEYQIGYICSGIFGD
wvsfncinleygigyicsgitgd NPRPNDKTGSCGPVSSNGANGV 19V nprpndktgscgpvssngangv , kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpel TLDCIVPCFWVELIRGRPKEN
tgidciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:66) NO:66) VKLACNSSLCPVSGWAPLSKDN
yklagnsslcpvsgwaPLskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFLTQGALLNDKHSNGT lecrtttltwailndkhsngt IKDRSPYRTLMSVPIGVVPSPY
ikdrspyrtimsVpigVvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnnilrtqesecacvng numbering:
NI-SCFTVMTDGPSNGQASYKIFRI 99P/ 9 scttvmtdgpsngclasykitri Ca109-EKGKIVKSVEMNAPNYHYEECS 6T/2 051 , ekgkivksvemnapnyhyeecs c155 165V
CYPDSSEITCVCRDNWHGSNR?
cypdsseitcvcrdnwhgsnrp S1 , WVSFNQNLEYQIGYICSGIFGD 100L/408M/4 wvsfnqnleycjigyicsgitgd NPRPNDKTGSCGPVSSNGANGV 19V nprpndktgscgpvssngangv , kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVEDIRGRPKEN
tgldciVpctwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:67) NO:67) VKLAGNSSLCDVSGWAPLSKDN Ni yklagnsslcpvsgwaPLskdn SVRIGSKGDVFVIREPFISCS? numbering: svrigskgdvfvirepfiscsp Ni- LECRTFFLTQGALLNDKHSNGT 99P/177I/19 lecrtffltqgallndkhsngt Ca109- IKDRSPYRTLMSVPIGAVPSPY 6T/205I, ikdrspyrt1msVpigAvpspy c155_S1 NARFESIAWSASACHDGINWLT 165A, nArfesIawsasachdginwit igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG 19V, tikswrnni1rtqesecacvng SOFTVMTDGPSNGOASYKIFRI 161V/172A scftvmtdgpsnggasykifri SUBSTITUTE SHEET (RULE 26) EKGKIVKSVEMNAPNYHYEECS
ekgkiyksyemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR?
cypdsseitcycrdnwhgsnrp WVSFNQNLEYQIGYICSGIFGD
wvsfnqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KCESFKYGNGVWIGRTKSISSR
kgfsfkygnqywigrtksissr NGFEMIWDPNGWIGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgstvMhpel TGLDCIVPCFWVELIRGRPKEN
tgidciVpcfwvelirgrpken TIWTSGSSISFCCVNSDTVOWS
tiwtsgssisfcgynsdtygws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:68) NO:68) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpysgwaPLskdn SVRIGSKGDVFVIREDFISCS?
syrigskgdvfvirepfiscgp LECRTFFIXQGALLNDKHSNGT
lecrtff1tqgailndkhsngt IKDRSPYRTLMSVPIGIVPSPY
ikdrspyrtimsVpigIvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAILKYNGIITD igiTgpdngayaIlkyngiitd Ni TIKSWRNNILRTQESECACVNG
tikswrnniirtqesecacvng numbering:
SOFTVMTDGPSNGQASYKIFRI Ni- 99P/177I/19 scftvmtdgpsngqasykifri EKGKIVKSVEMNAPNYHYEECS Ca109- 6T/2 051, ekgkiyksyemnapnyhyeecs CYPDSSEITCVCRDNWHGSNR?
cypdsseitcycrdnwhgsnrp c155 Si 1651, WVSENQNLEYQIGYICSGIFGD
wvstnqnleyqigyicsgitgd NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv 19V, KGFSFKYGNGVWIGRTKSISSR 161V/1721\
kgfsfkygngvwigrtksissr NGFEMIWDPNGWIGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgynsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:69) NO:69) VKLACNSSLCPVSGWAIYSKDN
vklagnsslcpvsgwaiyskcin SVRIGSKGDVFVIRERFISCS?
svrigskgdvfvirepfiscsp LECRTFFIXQGALLNDKHSNGT
lecrtff1tqga11ndkhsngt IKDRSPYRTLMSVPIGEVPSPY
ikdrspyrtimsVpigevpspy NARFESVAWSASACHDGINWLT
nArfesvawsasachdginwit IGISGEDNGAMAYLKYNGIITD
igiegpdngavavikyngiitd TIKSWRNNILRTQESECACVNG
tikswrnniirtgesecacvng SOFTVMTDGPSNGQASYKIFRI
scttvmtdgpsnggasykitri Ni- EKGKIVKSVEMNAPNYHYEECS Ni ekgkiyksyemnapnyhyeecs Cal09 C CYPDSSEITCVCRDNWHGSNR? numbering:
cypdsseitcvcrdnwhgsnrp ysK0 WVSENQNLEYQIGYICSGIFGD 161V/172A
wvsfnqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpyssngangv KGFSYKYGNGVWIGRTKSISSR
kgfsYkygngywigrtksissr NGFEMIWDPNGWIGTDNNESIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVQHPEL
gdivginewsgysgsfvqhpe1 TGLDCIRPCFWVELIRGRPKEN
tgldcirpcfwyelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftid(C (SEQ
ID
NO:70) NO:70) VKLAGNSSLCPVSGWAPLCKDN vklagnsslcpvsgwaPLCkdn Ni svrigskgdvfvirepfVscsp numbering:
LECRTFELTQGALLNDKHSNGT
lecrtffltqgatlndkhsngt Ni- 99P/1/7I/19 IKDRSPYRTLMSVPICSVPSPY
ikdrspyrt1msVpiCSvpspy Ca109- 6T/205I, NARVESIAWSASACHDGINWLT
nArVesIawsasachdginwit c155 S1 1653, 100L/408M/4 igiTgpdngavaIlkyngiitd TIKSWRNNILRTQESECACVNG
tikswrnni1rtgesecacvng 2V G164 19V, 161V/172A, scftymtdgpsnggasykifri EKGKIVKSVEMNAPNYHYEECS
3444V 101C/122V/1 ekgkivksvemnapnyhyeecs cypdsseitcvcrdnwhgsnrp WVSENQNLEYQIGYICSGIFGD wvsfnqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpnciktgscgpvssngangy SUBSTITUTE SHEET (RULE 26) KCFSFKYGNGVWIGRTKSISSR kgfsfkygnqvwigrtksissr NGFEMIWDPNGWIGTDNNESIK ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL qdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKEN tgldciVpcfwvelirgrpken TIWTSGSSIVFCGVNSDTVGWS tiwtsgssiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:/l) NO:/1) VKLAGNSSLCPVSGWAPLCKDN vklagnsslcpvsgwaPLCkdn SVRIGSKGDIFVIREPFVSCS? svrigskgdIfyirepfVscsp LECRTFFTQGALLNDKHSNGT lecrtifitqgallndkhsngt IKDRSPYRTLMSVPICSPPSPY ikdrspyrt1msVpiCSPpspy Ni NARVESIAWSASACHDGINWLT nArVesIawsasachdginwlt numbering:
Ni- IGITGDDNGAVAILKYNGIITD 99P/177I/19 igiTgpdngavaIlkyngiitd Ca109- TIKSWRNNILRTQESECACVNG tikswrnni1rtqesecacvng 6T/205I, c155 V1 SCFTVMTDGPSNGQASYKIFRI scftvmtdgpsngclasykifri 141 V16 EKGKIVKSVE 165S, MNAPNYHYEECS ekgkivksvemnapnyhyeecs 63' 3101 CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp 19V, C I122V WVSENQNLEYQIGYICSGIFGD wvsfnqnleyqigyicsgifgd 161V/172A, G164C NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv 1141/166P, F174V S KGFSFKYGNGVWIGRTKSISSR 101C/122V/1 kgfsfkygngvwigrtksissr ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL qdivginewsgysgstvMhpel TGLDCIVPCFWVELIRGRPKEN tgldciVpcfwvelirgrpken TIWTSGSSIVFCGVNSDTVGWS tiwtsgssiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:72) NO:72) VKLAGNSSLCPVSGWAPLCKDN vklagnsslcpvsgwaPLCkdn SVRIGSKGDVFVIREPFVSCS? svrigskgdvfvirepfVscsp LECRQFFLTQGALLNDKHSNGT lecrQffltqga11ndkhsngt IKDRSPYRTLMSVPICSVPSPY ikdrspyrtlmsVpiCSvpspy Ni NARVESIAWSASACHDGINWLT nArVesIawsasachdginwit numbering:
IGITGETNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd Ni- 99P/1771/19 TIKSWRNNILRTQESECACVNG tikswrnnilrtqesecacvng Ca109- 6T/205I, SCFTVMTDGPSNGQASYKIFRI scttvmtdgpsngqasykitri c155 Ti 1653, EKGKIVKSVEMNAPNYHYEECS ekgkivksvemnapnyhyeecs 3iQSi0 100L/408M/4 1C y122 CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnLp 19V, WVSENQNLEYQIGYICSGIFGD wvstnqnleyqigyicsgitgd V G164C 161V/172A, NPRPNDKTGSCGPVSSNGANGV nprpndktgscgpvssngangv F174V 131Q, ¨ KGFSFKYGNGVWIGRTKSISSR
S444V 101C/122V/1 kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNESIK ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL qdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKEN tgldciVpcfwvelirgrpken TIWTSGSSIVFCGVNSDTVGWS tiwtsgssiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:73) NO:73) VKLAGNSSLCPVSGWAPLCKDN vklagnsslcpvsgwaPLCkdn SVRIGSKGDIFVIREPFVSCS? svrigskgdIfyirepfVscsp Ni LECRQFFLTQGALLNDKHSNGT lecrQffltqgallndkhsngt Ni- IKDRSPYRTLMSVPICSPPSPY numbering: ikdrspyrtlmsVpiCSPpspy Cal 09- NARVESIAWSASACHDGINWLT
nArVesIawsasachdginwit 6T/2051, c155 V1 IGITGPDNGAVAILKYNGIITD igiTgpdngavaIlkyngiitd 165S, 141 V16 TIKSWRNNILRTQESECACVNG fikswrnnilrtqesecacvng 6P T131 SCFTVMTDGPSNGQASYKIFRI scftvmtdgpsnggasykifri 19V, Q S101C EKGKIVKSVEMNADNYHYEECS ekgkivksvemnapnyhyeecs 161V/172A, I122V CYPDSSEITCVCRDNWHGSNR? cypdsseitcvcrdnwhgsnrp 1141/166D, G164C F WVSFNQNLEYQIGYICSGIFGD wysfnqnleygigyicsgifgd 174V34 NPRPNDKTGSCGPVSSNGANGV 131Q, nprpndktgscgpvssngangv 44V KGFS7KYGNGVWIGRTKSISSR 64C/174V/44 kgfsfkygngvwigrtksissr NGFEMIWDPNGWIGTDNNESIK ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL qdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKEN tgldciVpcfwvelirgrpken SUBSTITUTE SHEET (RULE 26) TIWTSGSSIVFCGVNSDTVGWS
tiwtsgssiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:74) NO:74) VKLAGNSSLCPVSGWAPLCKDN
vklagnsslcpvsgwaPLCkdn SVRIGSKGDVFVIREPFISCSD
svrigskgdvfvirepfiscsp LECRMFFLTQGALLNDKHSNGT
lecrMttltqgailndkhsngt IKDRSPYRTLMSVPLCSVPSPY ikdrspyrt1msVpLCSvpspy NARVESIAWSASACHDGINWLT
nArVesIawsasachdginwlt numbering:
Ni- IGITGPDNGAVAILKYNGIITD 9 igiTgpdngavaIlkyngiitd Cal09- TIKSWRNNILRTQESECACVNG 6T/2051 tikswrnniIrtgesecacvng , c155 T1 SCFTVMTDGPSNGQASYKIFRI
softvmtdgpsnggasykifri 31M 116 EKGKIVKSVEMNAPNYHYEECS 165S, 100L/4 ekgkivksvemnapnyhyeecs M/4 cypdsseitcvcrdnwhgsnrp I S444A WVSENQNLEYQIGYICSGIFGD 19V, , wvsfngnleygigyicsgifgd S101C NPRPNDKTGSCGPVSSNGANGV 161V/1,2A, nprpndktgscgpvssngangv 42I/444A kgfsfkygngvwigrtksissr , ngfemiwdpngwtgtdnnfsik gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSIIAFCGVNSDTVGWS
tiwtsgsIiAfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:/5) NO:/5) VKLAGNSSLCPVSGWAPLCKDN
vklagnsslcpvsgwaPLCkdn SVRIGSKGDVEVIREPFVSCS?
svrigskgdvfvirepfVscsp LECRMFFLTQGALLNDKHSNGT
lecrMff1tqga11ndkhsngt IKDRSPYRTLMSVPICSVPSPY Ni ikdrspyrt1msVpiCSvpspy NARVESIAWSASACHDGINWLT
nArVesIawsasachdginwlt numbering:
Ni- ISITGPDNGAVAILKYNGITTD 992/1771/19 igiTgpdngavaIlkyngiitd Ca109- TIKSWRNNILRTQESECACVNG 6T/2 051 tikswrnni1rtqesecacvng c155 T1 SCFT , scftvmtdgpsnggasykifri , 31M S44 EKGKIVKSVEMNAPNYHYEECS 100L/408M/4 ekgkivksvemnapnyhyeecs 21 S101 CYPDSSEITCVCRDNWHG3NR?
cypdsseitcvcrdnwhgsnrp C T122V WVSENQNLEYQIGYICSGIFGD 19V, 161V/172 wvsfngnleygigyicsgifgd A, nprpndktgscgpvssngangv , F174V S KGFSFKYGNGVWIGRIKSISSR 101C/122V/1 kgfsfkygngvwigrtksissr 64c/ 174v/44 ngfemiwdpngwtgtdnnfsik gdivginewsgysgstvMhpel TGLDCIVPCFWVELIRGRPKEN
tgldciVpctwvelirgrpken TIWTSGSIIVFCGVNSDTVGWS
tiwtsgsIiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:76) NO:76) VKLAGNSSLCPVSGWAPLCKDN
vklagnsslcpvsgwaPLCkdn svrigskgdIfvirepfiscsp LECRMFFLTQGALLNDKHSNGT
lecrMffltqga1lndkhsngt IKDRSPYRTLMSVPLCSPPSPY Ni ikdrspyrt1msVpLCSPpspy Ni- NARVESIAWSASACHDGINWLT numbering: nArVesIawsasachdginwit Ca109- IGITGPDNGAVAILKYNGIITD 99P/1771/19 igiTgpdngavaIlkyngiitd c155 V1 TIKSWRNNILRTQESECACVNG 6T/205I, tikswrnni1rtgesecacvng 141 V16 SCFTVMTDGPSNGQASYKIFRI 165S, scftvmtdgpsnggasykifri 6? T131 EKGKIVKSVEMNAPNYHYEECS 100L/4 08M/4 ekgkivksvemnapnyhyeecs M I163L CYPDSSEITCVCRDNWHGSNR? 19V, cypdsseitcvcrdnwhgsnrp S442I WVSENQNLEYQIGYICSGIFGD 161V/172A, wvsfngnleygigyicsgifgd S444A S NPRPNDKTGSCGPVSSNGANGV 1141/166P, nprpndktgscgpvssngangv 1016_01 KGFSFKYGNGVWIGRTKSISSR 131M/163L/4 kgfsfkygngvwigrtksissr 640 F17 NGFEMIWDPNGWIGTDNNFSIK 42I/444A, ngfemiwdpngwtgtdnnfsik gdivginewsgysgsfvMhpel tgldciVpcfwvelirgrpken TIWTSGSIIAFCGVNSDTVGWS
tiwtsgsIiAfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:77) NO:77) SUBSTITUTE SHEET (RULE 26) VKLAGNSSLCPVSGWAPLCKDN
vklagnsslcpvsgwaPLCkdn svrigskgdIfyirepfVscsp LECRMFFLTQGALLNDKHSNGT
lecrMffltqga1lndkhsngt IKDRSPYRTLMSVPICSPPSPY Ni ikdrspyrt1msVpiCSPpspy Ni- NARVESIAWSASACHDGINWLT numbering: nArVesIawsasachdginwit Ca109- IGITGPDNGAVAILKYNG1ITD 99P/1771/19 igiTgpdngavaIlkyngiitd c155 V1 TIKSWRNNILRTQESECACVNG 6T/205I, tikswrnniirtqesecacvng 141 V16 SOFTVMTDGPSNGQASYKIFRI 1653, scftvmtdgpsnggasykifri 6P_T131 EKGKIVKSVEMNAPNYHYEECS 100L/4 08M/4 ekgkivksvemnapnyhyeecs M S442I CYPDSSEITCVCRDNWHGSNR2 19V, cypdsseitcvcrdnwhgsnrp S101C WVSFNQNLEYQIGYICSGIFGD 161V/172A, wvsfnqnleyqigyicsgifgd I122V G NPRPNDKTGSCGPVSSNGANGV 1141/166P, nprpndktgscgpvssngangv 164C Fl KCFSFKYGNGVWIGRTKSISSR 131M/4421, kgfsfkygngvwigrtksissr 74V_544 NGFEMIWDPNGWTGTDNNFSIK 101C/122V/1 ngfemiwdpngwtgtdnnfsik qdivginewsgysgsfvMhpel tgldciVpcfwvelirgrpken TIWTSGSIIVFCGVNSDTVGWS
tiwtsgsIiVfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:78) NO:78) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskcin SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFLTQGALLNDKHSNGT
lecrttfltqgailndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrt1msVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAMAYLKYNGIITD
igiTgpdngavavikyngiitd TIKSWPNNILRTQESECACVNG Ni tikswrnni1rtqesecacvng Ni- SCFTVMTDGPSNGQASYKIFRI numbering:
scftumtdgpsngclasykifri Ca109- EKGKIVKSVEMNAPNYHYEECS 99P/1771/19 ekgkivksvemnapnyhyeecs cl30_T4 CYPDSSEITCVCRDNWHGSNR? 6T, 165S, cypdsseitcvcrdnwhgsnrp 53V 120 WVSENQNLEYQIGYICSGIFGD 100L/408M/4 wvsfnqnleygigyicsgifgd 5V NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
qdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgldciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdcae1pftidk (SEQ ID
NO:87) NO:87) VKLAGNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskdn SVRIGSKGDVFVIREPFISCS?
svrigskgdvfvirepfiscsp LECRTFFLTQGALLNDKHSNGT
lecrtffltqga1lndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrtimsVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDSGAVAVLKYNGIITD
igiTgpdsgavavlkyngiitd TIKSWRNNILRTQESECACVNG Ni tikswrnni1rtqesecacvng SCFTIMTDGPSDGQASYKIFRI numbering: scftimtdgpsdgelasykifri Mil5 cl EKGKIIKSVEMKAPNYHYEECS 99P/1771/19 ekgkiiksvemkapnyhyeecs 55 1205 CYPDSSEITCVCRDNWHGSNR2 6T, 165S, cypdsseitcvcrdnwhgsnrp wvsfnqnleyqmgyicsgvfgd NPRPNDKTGSCGPVSSNGANGV 19V, nprpndktgscgpvssngangv kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
qdivginewsgysgsfvMhpe1 TCLDCIVPCFWVELIRGRPEEN
tgldciVpctwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:88) NO:88) Ni- VKLAGNSSLCPVSGWAPLSKDN Ni yklagnsslcpvsgwaPLskdn M115_c1 SVRIGSKGDVFVIREPFISCS2 numbering: svrigskgdvfvirepfiscsp 55 T131 LECRQFFLTQGALLNDKHSNGT 99P/177I/19 lecrQffltqgallndkhsngt 0_1205V IKDRSPYRTLMSVPIGSVPSPY 6T, 165S, ikdrspyrt1msVpigSvpspy SUBSTITUTE SHEET (RULE 26) NARFESLAWSASACHDGINWLT 100L/4 08M/4 nArfesTawsasachdginwlt IGITGPDSGAVAVLKYNGIITD 19V, igiTgpdsgavavlkyngiitd TIKSWRNNILRTQFSECACVNG 161V/172A, tikswrnni1rtqesecacvng scftimtdgpsdomasykifri EKGKIIKSVEMKAPNYHYEECS
ekgkiiksvemkapnyhyeecs cypdsseitcvcrdnwhgsnrp WVSFNQNLFYQMGYICSGVFGD
wvstricinleyqmgyicsgvtgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv KOFSFKYGNOVWIGRTKSISSR
kgfsfkygngvwigrtksissr KGFEMIWDPNGWTGTDNKFSIK
kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
qdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaeipftidk (SEQ ID
NO:89) NO:89) VKLAGNSSLCPVSGWAPLAKDN
vklagnssicpvsgwaPLAkdn SVRIGSKGDVFVIREPFISCSP
svrigskgdvfvirepfiscsp LECRMFFL,TQGALLNDKHSNGT
lecrMfflteiga11ndkhsngt ikdrspyrt1msVpLgSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit Ni-IGITGPDSGAVAVLKYNGIITD
igiTgpdsgavavlkyngiitd numbering:
Mil5 cl TIKSWRNNILRTQESECACVNG
tikswrnniirtgesecacvng 6T, 165S,1/408M/4 scftimtdgpsdgclasykitri EKGKIIKSVEMKAPNYHYEECS
ekgkiiksvemkapnyhyeecs CYPDSSEITCVCRDNWHGSNR? 19V
cypdsseitcvcrdnwhgsnrp , S442I t73 WVSFNQNLEYQMGYICSGVFGD 161V/172A
wvsfncinleycimgyicsgvtgd , 444A 12 NPRPNDKTGSCGPVSSNGANGV 101A/131M/1 nprpndktgscgpvssngangv KGFSFKYGNGVWIGRTKSISSR 63L/442I/44 kgfsfkygngvwigrtksissr KGFEMINDPNGWIGTDNKFSIK kgfemiwdpngwtgtdnkfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVFLIRGRPEEN
tgldciVpcfwvelirgrpeen TIWTSGSIIAFCGVNSDTVGWS
tiwtsgsIiAfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:90) NO:90) VKLACNSSLCPINGWAPLSKDN
vklagnsslcpingwaPLskdn SVRIGSKGDVFVIRERFISCSH
sVrigskgdvfvirepfiscsh LECRQFFITQGALLNDKHSNGT
lecrQttlteigailndkhsngt VKDRSPHRTLMSVPIGSVPSPY
vkdrsphrtimsVpIgSVpspy NARFESIAWSASACHDGTSWLT
nArfesIawsasachdgtsw1t IGITGPDNGAVAVLKYNGIITD Ni igiTgpdngavavikyngiitd Ni- TIKSWRNNILRTQESECACVNG numbering: tikswrnnilrtqesecacvng V5T04 c1 SCFTVMIDGPSNGQASYKIFKN 99P/177I/19 scttvmtdgpsngclasykitkm 55 1106 EKGKVVKSVELDAPNYHYEECS 6T, 165S, ekgkvvksveldapnyhyeecs V T131Q CYPNAGEITCVCRDNWHGSNR? 100L/4 08M/4 cypnageitcvcrdnwhgsnrp V163I WVSFNQNLEYQIGYICSGVFGD 19V, wvsfnqnleycjigyicsgvtgd A166V_I NPRPNDGTGSCGPVSSNGAYGV 161V/172A, nprpndgtgscgpvssngaygv 205V KGFSFKYGNGVWIGRTKSTNSR 131Q, 106V, kgfsfkygngvwigrtkstnsr SGFEMIWDPNGWIETDSSFSVK 1631, 166V
sgfemiwdpngwtetdssfsvk QDIVAITDWSGYSGSFVMHPEL
gdivaitdwsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKES
tgldciVpctwvelirgrpkes TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:91) NO:91) Ni-VILIGNSSLCDIRGWADLSKDN Ni viltgnssicpirgwaDLskdn SVRIGSKGDVFVIREPFISCSH numbering: SVrigskgdvfvirepfiscsh WSN33¨c 155 G10 LECRTFFDTQGALLNDKHSRGT 99P/177I/19 lecrtffltqga1lndkhsrgt 5S 1106 FKDRSPYRTLMSVPIGSVPSPY 6T, 165S, fkdrspyrT1msVpIgSVpspy NRFESIAWSASACHDGMGWLT 100L/408M/4 nArfesIawsasachdgmgw1t IGITGPDDUAVAVLKYNGIITE 19V, igiTgpddgavavlkynGiite ¨V163I¨ TIKSWRKNILRTQFSECTCVNG 161V/172A, tikswrkni1rtqesectcvng A166V¨R SCFTIMIDGPSDGLASYKIFKI 1055, 106V, scftimtdgpsdglasykifki SUBSTITUTE SHEET (RULE 26) 210512 EKGKVTKSIELNAPNSHYEECS 157T, 1631, ekgkvtksielnapnshyeecs 05V CYPDTGKVMCVCRDNWHGSNR2 166V, 210G
cypdtgkvmcvcrdnwhgsnrp WVSFDQNLDYKIGYICSGVFGD
wvsfdqn1dykigyicsgvfgd NPRPKDGTGSCGPVSADGANGV
nprpkdgtgscgpvsadgangv KCFSYKYGNGVWIGRTKSDSSR
kgfsykygngvwigrtksdssr HGFEMIWDPNGWTETDSRFSMR
hgfemiwdpngwtetdsrfsmr QDVVAITNRSGYSGSFVMHPEL
gdvvaitnrsgysgstvMhpel TGLDCMVPCFWVELIRGLPEED
tgldcmVpcfwvelirgipeed AIWTSGSIISFCGVNSDTVDWS
aiwtsgsiisfcgvnsdtvdws WPDGAELPFT1DK (SEQ ID wpdgae1pftidk (SEQ ID
NO:92) NO:92) VILTGNSSLCPIRGWAPLSKDN
viltgnsslcpirgwaPLskdn SVRIGSKGDVFVIREDFISCSH
SVrigskgdyfyirepfiscsh LECRQFFLTQGALLNDKHSRGT
lecrQff1tqga11ndkhsrgt FKDRSPYRTLMSVPIGSVPSPY
fkdrspyrT1msVpIgSVpspy Ni-NARFESIAWSASACHDGMGWLT Ni nArresIawsasachdgmgw1t IGITGPDDGAVAVLKYNGIITE numbering: igiTgpddgavaylkynGlite c 155 Gy0 TIKSWRKNILRTQESECTCVNG 992/1771/19 tikswrkni1rtqesectcvng 1106 SCFTIMTDGPSDGLASYKIFKI 6T, 165S, scftimtdgpsdglasykifki VS T131Q EKGKVTKSIELNAPNSHYEECS 100L/408M/4 ekgkvtksielnapnshyeecs A157T CYPDTGKVMCVCRDNWHGSNR? 19V, cypdtgkvmcvcrdnwhgsnrp V1631 A WVSFDQNLDYKIGYICSGVEGD 161V/112A, wvsfdqnidykigyjcsgvfgd 166V R2 NPRPKDGTGSCGPVSADGANGV 131Q, 105S, nprpkdgtgscgpvsadgangv KGESYKYGNGVWIGRTKSDS52 106V, 157T, kgfsykygngvwigrtksdssr 5V 120 HGFEMIWDPNGWTETDSRFSMR 1631, 166V, hgfemiwdpngwtetdsrfsmr gdvvaitnrsgysgsfvMhpe1 TGLDCMVPCFWVELIRGLPEED
tgldcmVpcfwvelirglpeed AIWTSGSIISFCGVNSDTVDWS
aiwtsgsiisfcgynsdtvdws WPDGAELPFTIDK (SEQ ID wpdgae1pftidk (SEQ ID
NO:93) NO:93) VKLACNSSLCPVSGWAPLSKDN
vklagnsslcpvsgwaPLskcin SVRIGSKGDVFVIREPFISCS?
svrigskgdyfyirepfiscsp LECRQFFLTQGALLNDKHSNGT
lecrQff1tqga11ndkhsngt IKDRSPYRTLMSVPIGSVPSPY
ikdrspyrtimsVpigSvpspy NARFESIAWSASACHDGINWLT
nArfesIawsasachdginwit IGITGPDNGAVAVLKYNGIITD igiTgpdngavavikyngiitd TIKSWRNNILRTQESECACVNG
tikswrnniirtqesecacvng numbering:
Ni- SCFTVMTDGPSNGQASYKIFRI
scttvmtdgpsngqasykifri Ca109- EKGKIVKSVEMNAPNYHYEECS 6T 165S
ekgkivksvemnapnyhyeecs , , c155 T1 CYPDSSEITCVCRDNWHGSNR? 100L/4 08M/4 cypdsseitcvcrdnwhgsnrp wvsfnqnleyqigyicsgifgd , bV NPRPNDKTGSCGPVSSNGANGV 161V/112A
nprpndktgscgpvssngangv 131Q , KGFSFKYGNGVWIGRTKSISSR kgfsfkygngvwigrtksissr NGFEMIWDPNGWTGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
gdivginewsgysgsfvMhpe1 TGLDCIVPCFWVELIRGRPKEN
tgidciVpcfwvelirgrpken TIWTSGSSISFCGVNSDTVGWS
tiwtsgssisfcgvnsdtvgws W2DGAELPFTIDK (SEQ ID wpdgae1pftid(C (SEQ
ID
NO:94) NO:94) VKLAGNSSLCPVSGWAPLAKDN
vklagnsslcpysgwaPLAkdn SVRIGSKGDVFVIREPFISCS2 Ni svrigskgdvfvirepfiscsp Ni- LECRMFFLTQGALLNDKHSNGT numbering: lecrMffltqga1lndkhsngt Ca109- IKDRSPYRTLMSVPLGSVPSPY 992/1771/19 ikdrspyrt1msVpLgSypspy c155_S1 NARFESIAWSASACHDGINWLT 6T, 165S, nArresIawsasachdginwit 01A T13 IGITGPDNGAVAVLKYNGIITD 100L/4 08M/4 igiTgpdngavavlkyngiitd 1M1163 TIKSWRNNILRTQESECACVNG 19V, tikswrnni1rtqesecacvng L ,4421 SCFTVMTDGPSNGQASYKIFRI 161V/172A, scftvmtdgpsngqasykifri 5444A EKGKIVKSVEMNAPNYHYEECS 101A/131M/1 ekgkivksvemnapnyhyeecs 1205V CYPDSSEITCVCRDNWHGSNR2 63L/4421/44 cypdsseitcvcrdnwhgsnrp wvsfnqnleyqigyicsgifgd NPRPNDKTGSCGPVSSNGANGV
nprpndktgscgpvssngangv SUBSTITUTE SHEET (RULE 26) KCFSFKYGNGVWIGRTKSISSR
kqfsfkyqnqvwiqrtksissr NGFEMIWDPNGWIGTDNNFSIK
ngfemiwdpngwtgtdnnfsik QDIVGINEWSGYSGSFVMHPEL
qdivginewsgysgsfvMhpel TGLDCIVPCFWVELIRGRPKEN
tqldciVpcfwvelirgrpken TIWTSGSIIAFCGVNSDTVGWS
tiwtscssIiAfcqvnsdtvgws WPDGAELPFTIDK (SEQ ID wpdgaelpftidk (SEQ ID
NO:95) NO:95) SUBSTITUTE SHEET (RULE 26) 1.00C/1.63C;.
100016301.73V;
100C,1163CII73V445V;
130Q/4431;
100A/162L/445V;
130M/4431;
100A/130M/162L/4431/445A;
130M/162L/4431/445A;
1761/2041;
100C/12IV/163C/173V/445V; and/or 12IV/445V.
.36. The polypeptide of any one of claim 32-34, wherein the polypeptide include throe or More of the listed amino acid residues relative - to .SEQ.- ID N-0.14 when aligned by protocol or prottieol.2-.
37. The polypeptide of any one tifelaint 32-34, wherein the polypeptide includes five or more of the listed amino acid residues relative to SEQID NO A when aligned by protocol I
or protocol 2.
38. The polypeptide of any one of claini.-32-34,.wherein.the polypeptide.include* Seven . or more of the listed. amind acid residues relative lo ,SEQ ID NO when aligned by protocol I
=or protocol.
39.. The polypeptide of any one of claim 32.-34, wherein the. polypeptid.ci includes nine or.
mire of the listed amitIQ acid.. reSiduca relative to.SEQ ID NO ,A.. when aligned by protocol I
or protocol 2.
40. The it:on-naturally cieentrintrpo/Y0Ptidoofelaint 1., wherein, the polypeptide is at 30. least 75%, 80%, 85%, 90%, 91%,- 92%, 93%, 94%,. 95%,.96%, 97%, 98%,.or-99% identical to the N4 NA polYpeptide of SEQ1D.N0:4, and wherein the non-naturally occurring.
polypeptide includes I, or both of the following amino acid residues relative to SEQ 11).-NOA.
when aligned. byprotocol 1 or protocol 2: I.64Q/E, I95S.

41.. The .ion-naturally occurring pOlypoptido of Claim.). 1, Wherein the peilypopilde is ful at least 75%, 80%, .85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%õ 97%, 98%, Or 99% identical to the N5 NA.polypeptide Of SEQ1D.N0:5., and Wherein the tion4latervilly occurring polypeptide includes 1, .2.3. 4 5.6. 7, 8õ9, 10, I 1, 12, or all 13 oldie tbllowing amino acid residues=relative to SEQ ID NO:S.wben aligned by protocol 1 or protocol 97L, 410M, 901),98A,1001,192SIA, 110D, 128Q/M, 1601, 162V/A/1, 163 VIP, 193Q/T, 4451; .or 01 at least 75%õ..80%, :85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ .1D NO:5, and wherein the non-naturally 19 ()Courting- pOlypeptide includes 2, 3, 4, 5, 6, 7, S, 9, 10õ 11õ12õ 13, 14, 15., Or all 16 Of the following amino .aeid residues relatiye: to SEQ. IP ND:5 when aligned hyproiocci 1 or protocol 2:
971.,, 410M, 96P, W4A.,100N, 102S/A., 1.0W, 110D, 128Q/M, 154T, 1601, 162V/A/LT, 163V/P, 1741, 193Qir,. 4451.
42. The polypeptide of clann41, wherein the poIypeptide is (a). at least 75%, 80 ,4, 85%, 90%, 91%, 92%, 93%,.. 94%, 95%, 96%,. 97%, 98%, o9% idoticgl to. dle.N5 NA polypeptide of SEQ ID NO:5,. and Wherein the non-naturally occurring pohy. peptide includes 1, 2, 3, 4õ 5, k 7, S-õ 9,. 10, 11, 12, or all 1.3 of *Mowing .20 amino acid residuesTelativeloSEQIDNO:5 when aligned by protocol 1 or protocol 2:
(i) 9M, 410M, 96p,. 9.8A, 1001s!,. 102S/A, 110D, 128Q/M, 1601, 162V/A/1, 163 V/P, 1930'T, 445L or (ii) 96P, 98A, 100N,102SIA, 11:01), 128Q/M, 1601, 162V/A/1, 163 VIP, 193Q/T, 4451.
2.5 43.õ The polypepride of' claim 41, wherein the poiypeptide is (h) at least 75%, 80%,. 85%, :90%, 91%, 92%, 93%, 94%, 95%, 90%, 97%, 98%, or 99% identical to the N5 NA. polypeptide of SEQ ID NO:5õ and Whcrein.the non-naturally occuttina polypeptide. includes 2, 3, 4, 5, .6, 7, 8õ9õ .10õ. 11, 12, 13, 14, 15, or all 16, ofthe 30. following amino acid residuts: relative:to SEQ. ID NO:5 when aligned by protocol 1 or omtocial 2.
.(i) 97L 410M, 96P, 98A, 100N,. 1.02$/A, 1Ã13Y 110D, 128Q1M, 154T, 1601, 162V/ATIIT, 163 VIP, 1741, 193Q/T, 4451; or :(ii) 96P, 98A, MN, 102S/A, 1.03V, 110D, 128Q/M, 1541, 1601, 162V/A/1/T, 163V/P, 1741, 193Q1I, 4451.
44. The polypeptide of any= one..of taim 41-43,.whereiri the polypepOde includes one or more of the ii)ilowingsets'of.ainino:Acid rosidoes: relative to SEQ ID. NO:5 when aligned by protocol 1 or protocol 2:
(i) 97L/410M;
100N/102A;
98C/161C;
128Q/445I;
128M/445I;
98A/1.2 8K/44.51447A
971.198A/1 28M,445.1447A;
5 1.28M1451/447A;
-97L/ 2M/4451/447A;

1111.1.0313c 6P.1.1931,2021;
.20 96P/1.741193T; andior 96P/1741/193T/2021; or 100N/102A;
98C/16IC;
25 128Q/445I;
128M/445I;
98A/128M/4451/447A;
971198A1128M144511447A.;
1 28M144511447.A;
30. 97.1.11 281W4451/447 A;
96P/1931;
1111/163P;
96P/1931/2021;
96P/1741/1 93T; and/or

7]

45. The polypeptide of any one of claim 41-43, wherein the polypeptide includes three or more of the listed amino acid residues relative to SEQ ID 3.'40:5 when aliened by protocol I
or protocol 2.
46. The polypeptide of any one of claim 41-43, wherein the polypeptide includes five or more of the listed. amino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2.
47. The polypeptide of any one of claim 41-43, wherein the polypeptide includes seven or more of the listed amino acid residues relative to SEQ ID NO:5 when aligned by protocol I
or protocol 2, 48. The .polypeptide of any one of claim 4.1.43, wherein the polypeptide includes nine or more of the listed amino acid residues relative to SEQ ID NO:5 when aliened by protocol .1 or protocol 2.
49. The non-naturally occurring po.lypeptide of claim I, wherein the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N5 NA polypeptide of SEQ tD NO:5, and wherein the non-naturally occurring polypeptide includes 1, or both of the following amino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2: 162 Q/E, 200S.
50.. The non-naturally occurring polypeptide of claim I. wherein the polypeptide is (a) at feast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N6 NA polypeptide of SEQ ID NO:6, and wherein the non-naturally occurring polypeptide includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or all IL of the .1ollowing amino acid residues relative to SEQ ID NO:6 when aligned by protocol I or protocol 2:
9(.P, 103.N, 113D, 13IQ, 1611, 162P. 1631, 165S/TIVIA, 196Q, 203Y. 445S; or (h) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N6 NA polypeptide of SEQ ED NO:6, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following amino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2:
99P, 103N, 1.05S, IO6V, .1131), 131Q. :157T, 1611, 162P, 1631/4 I65SrUVIA/I, 166V1P, 196Q, 203Y, 445S.
Si. The polypeptide of ci4ipi 50, wheroin the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N6 NA polypeptide of SEQ: ID NQ:6, and ',wherein the non-naturally otxurring polypeptide includes 1, 2, 3.4.,. 5, 6., 7, 8,9, .10, or all 11 of the following ainino 119 arid reSidues relatrve to SEQ ID NQ:6 when aligned by protocol 1 Or protocol 2:-99P, MN, -31), 1611. 1621', 1631, J.65SIT(V/A, 196Q, 203Y, 445S.
52 The polypeptide of Claiin50. Wherein the polypeptide .(hl At least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N6. NA polypeptide of SEQ ID NO:6, and Wherein die non-naturally occurring polypeptide hielades 2, 3, 4, 5, 6, 7, 3, 9, 10, 11, 12, 13, 14, or all 15 of the followingentinozeid residnes relative to. SEQ ID NO;6 When alitried by protocol 1 bt-protocol 2:
99P, 103N, 105S, 106V, 113D, 131Q, 157T, 1611, 162P, 1631/L, 165S/T/V/A/I, 166V/P, 196Q, 203Y, 445S.
53, The polypwtide of any &e1aim 50-52, wherein the oOksipePtide includes one Or More of the following:sets of aminO acid residues: relatiVe to SEQ ID NO:6 When aligned by protocol I or protocol 2:
101C/164C;
101C/164C/174V;
101C/164C/174V/447V, 122V1447. V;
101.A116314 99W196T; And/or 99P/196T/205I.

54. The polypeptide of any one of claim 50-52, Wherein the polypeptide includes three or more of the listed amino acid residues relative to SEQ1D NO:6 when aligned by protocol I
or protocol 2.
55. The polypeptide of any one of claim 50-52, wherein the polypeptide includes five or more of the listed aminoneid residues relative to SEQ ID NO:6 when aligned byprotocol I
or protocol 2.
56. The polypeptide of any one of claim 50-52, wherein the polypeptide includes seven or more of the listed amino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2.
57. The polypeptide of any one of claim 50-52, wherein the polypeptide includes nine or more of the listed amino acid residues relative to SEQ ID NO:6 when aligned by protocol .1 or protocol 2.
58. The non-naturally occurring polypeptide of claim 1, wherein the polypeptide is at.
least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N6 NA polypeptideof SEQ ID NO:6, and wherein the non-naturally occurring polypeptide includes a 165E amino acid mutation relative to SEQ ID NO:6 when aligned by protocol I. and optionally also includes a 177V amino acid mutation relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2.
59. The non-naturally occurring polypeptide of claim 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 9.1%, 92%., 93%, 94%, 95%., 96%, 97%, 98%., or 99% identical to the N7 NA polypeptide of SEQ. ID NO:7, and Wherein the non-naturally occurring polypeptide includes 1, 2, 3,4. 5, 6, 7, 8, 9, 10, or all II of the following amino acid residues relative to SEQ ID NO:7 when aligned by protocol I Or protocol 2:
981?, 102N, 104S, 1121), 130Q, 1561, 1.621, 164V/AIL, I65V, 202Y, 448V; or (b) at least 75%, 80%, 85%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%. 98%, or 99% identical to the N7 NA polypeptide of SEQ ID NO:7, and wherein the non-naturally occurring .polypeptide includes 2, 3,4, 5, 6, 7, 8, 9, .10, 11, 12, or all 13 of the following amino acid residues relative to SEQ ID NO:7 when aligned by protocol I or protocol 2:
98P, 102N, 104S, 1121), 130Q, 1561, 1621, 164WA/I, I65V, .1761, 202Y, 4461, 448V.

60. The potypeptide Of claim 59,. wherein the poiypeptide.is fol at least 75%, 80%, .85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%õ 97%, 98%, or 99% identical to the N7 NA.polypeptide Of SEQ
and Wherein the non4niturany occurring polypeptide includes 1, 3, 4, 5:, 6, 7, 8õ9, IQ, trail ii of the followingatnino acid residues relative. to .SEQ ID N.O.:7 when aligned by, protocol I or protocol .2!
98P, 102N,1045, 112), 130Q. 156T, 1621, 164V/A/1:, 165V, 202Y, 448V.
61. The polypeptide of slairrt 59õ wherein the polypeptide is (b) at least 75%, 80%, 85%, .90%, 91%, 92%, 93%, 94%,. 95%, 96%, 97%, 98%, or 99% identical to the N7. NA pnlypeptide of SEQ1D N.0:7, and wherein the non-naturally .ocentring.polypepOde, includes 2, 3. 4, .5, 7, 8, 9, 10, 11., 1.2, or 411 13 of the following amino. acid rosidumrCiativc to SEQ. ID NO:7 when aligned hy.protocol 1 or protocol 2:.
98P, 102N, 104S, 1121), 130Q, .1561. .1621, 164V/AIL 165V, 1761, 2112Y, 4461, 448V..
5 62. The .polypeptide of any one of 'claim 59-61, wherein the poly-peptide includes one or there ofthe -following sets: of amino. aci d residues. relative lb SEQ NO: 7-When aligned by protocol .I Or protocol .2:
100C1163C;
100C/163C/173V;
100C/163C/173V/448V;
.991.,/446.11448A;
98.11(195T-, 446I/448A;
9p/ 95T/2041;
98P11.761/195T.; and/or 98P/1.761(195T12041, 63: The polypeptide of any claim; 59-61. Wherein the .pOlype pti de includes date; ot 30. ntOfe.of thelisted aminOneid residues relative:to SEQ IT) NO:7.when aligned byttrotoeol 1 or protocol 2.

64. The polypeptide of any one of claim 59 6.l,.wherein the poly-peptide includes five or more of the listed amino acid residues relative. to SEQ ID. NO:7 when aligned by protoCol 1 or protocol 2.
65. The polypeptide of any one of claim 596i.. wherein thopolypeptide includes seven or more of the listed amino aohl residues relative to SEQ fp NO:7 when aligned by protocol or protocol 2.
66. The polypeptide of any one of elaith 59-61, Wherein the polypeptide includes nine.Or more of the listed amino acid. residues relative to SEQ ID NO:7 when aligned by protocol 1 or protocol 2.
671 The no naturally occurring polypeptide of claim 1, wherein the polypeptide is at least 75,.:8()% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, *1%, or 99%
identical to the N7 NA polypeptide of SEQ. NO:7, and wherein the non-naturally occurring polypeptide includes one or both of the :following amino acid mutation relative to SEQ ID
NO:7 when aligned by protocol 1 or protocol 2: 164QIE, 1955:
OK, The Don-naturally occurring polypeptidie of chnin 1, wherein the polypeptide is 28 (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally Ocetnting polypeptidc ind ticks 1, 2,3 4, 5, 67, 8, 9, 10, or all II of the following amino acid reltidueS relative to SEQ ID NiCY8 When aligned by protocol 1 or protocol 2;
408M, 101N, 103A/S, 111D, 129Q, 16113.1., 163SITAI/A11, 164Võ 194Q, 201.Y, 4421; Or (b) at least 75%, 80%, 85".4, 90%, 91%, 92%; 93%, 94%, 95%; 96%* 97%, 98%;
or 99% identical to the N8 NA polypeptide of $EQ, ID NE).:.8, and wherein the non-naturally oecurringpolypeptide includcs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 Of the following amino acid residues relative tO SEQ 113-NO:8 when aligned by protocol 1 or protocol 2:
981,, 408M. 'WIN, 1.03A/S. 104V, 111D. 129Q/M., 163SIT4'IA/I/SiT, 164 V/P, 1751.
194Q, 201Y, 2031, 4421 69. The poly-peptide of claim 68, wherein the poiypeptide is (a) at least 75%, 80%, .85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA poly-peptide Of SEQ ID NO:8, and Wherein the noti4uantally occurring polypeptide includes 1, 2, 3, 4, 5:, 6, 7, 9,10, or all 1.1 of the following amino acid residues:relative t.o SEQ ID NO:8 when aligned by: protocol I or Kowa') 2!
(i) 408M, MIN, IONS, ii ID, 129Q, 160P, 161L, 163S/T/V/All, 164V, 194Q, 201Y,.4421; or (ii) 101N, 103A/S, 111D, I 29Q, 160P, 161L, 163S/1-IV/A/I, 164V, 194Q, 201Y, 4421.
70. The polypeptide of claim 68, wherein the polypeptide is (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical: to the N8 NA :polypeptide SEQ
And Wherein the non-naturally pc-Outfit* polypeptide includ6 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, or all 15 of the following amino acid residues :relative to SEQ ID NO:8 when aligned by protocol 1 or protocol 2:
98L, 408M, 101N, 103A/S, 104V, 111D, 129Q/M, 160P, 161L, 163S/T/WANSIT, I64V/.1' 1751, 194Q, 201Y, 2031, 4421 or 011', 104V, 1111), 129Q/M, 1,00e, 61L, 1635,a7V/A.I/S(17, 1.64V/P; 1751, 194Q, 201rY 2031, 4421 .7j The. polypwtide of any laim 68-70, wherein the pOlYpePtide includes one Or Moro of the following:t;ets of amino acid residues: relative to SEQ ID NO:8 When aligned by protocol I or protocol 2:
(i) 98L/408M;
160P/163S;
101N/103A.;
990-162C;
990167.C11 72V;
129 Q/4421;
99A1161L;
99A/1611_14421;
161L/4421;

129M/4421;
99A/1291WI 611A421.1,114A;
981_199A/129M/16 IL/4421/444A
1 29Mil 61 I.14421.14.11A;
98L/129N111 6 II .14421/444A ; and'or 1751/2031; or (ii) 160P/163S;
101N/103A;
99C/162C;
99C/162C/172V;
129Q/442I;
99A/161L;
99A/161114421;
161L/4421;

99A/129M/161L/4421/444A;
98L/99Al1 29M/161114421/444A;
129M/1 OIL/4421/444A;
28 9$1,1129WIOIL/4421/111A; and/or 1 75112031.
72. The polypeptide of .any one of claim 68-70, wherein the polypeptide includeS three or more of the listed amine acid residues relative to SEQ ID NO; when allailed by protocol I
or protocol 73. The pplypeptide (:}1 any one of claim 6g-7Q, wherein the polypeptide .includes five or more of the listed amino aid residues relative to SEQ ID NO:a When aligned by protocol or protocol 2.
74.. The polypeptidc of any one of claim 6a-70, wherein the polypeptide includes seven or more of the listed amino acid residues relative to SEQ ID .NQ:8 when aliened by protocol I
or protocol 2.

75. The polypeptide of any one of claim 68-70, Wherein the polypeptide includes nine or more of the listed amino acid residues relative to SEQ1D NO:8 when aligned by protocol I
or protocol 2.
.76. The non-naturally occurring polypeptide of any one of claims 80-85, wherein the polypeptide comprises the amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9714 98%, or 99% identical to the amino acid selected from the group consisting of N8 mutants listed. in Table I (SEQ NO:35-38), when aligned by protocol 1, wherein the polypeptide includes all of the residues listed in Table 1 for an individual N8 mutant listed in Table I
77. The non-naturally occurring polypeptide of claim 1, wherein the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally occurring .polypeptide includes a 163E amino acid mutation relative to SEQ TD NO:8 when aligned by protocol I. and further may optionally include a 1945 mutation relative to SEQ
ID NO:8 when aligned by protocol 1 or protocol 2.
78. The non-naturally occurring polypeptide of claim 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 90% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of the following amino acid residues relative to SEQ ID NO:9 when aligned by protocol 1 .or protocol 2:
94P, 951., 1005, 126Q/M, 160V/A/I, 16IV, 191Q, 198Y, 439S, 441V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 of the following amino acid residues relative-to SEQ 1D N0:9 when aligned by protocol 1 or protocol 2:
941?, 951., 98N, ItX)SIA, 126Q/M, 152T, 1581, 160VIAMIT, 16IV, 191Q, I98Y, 2001, 4395, 441V.
79. The polypeptide of claim 78, wherein the polypeptide is (a) at least 75%, 80%, .8.5%,. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring poly-peptide includes I, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of the following arnino acid residues relative to SEQ ID NO9 when abated by protocol I or protocol 2;
9411., 95L., 1005, 126Q/M, 161V, 191Q, 198Y, 4395, 441V, 80_ The polypeptideof claim 78, wherein the polypeptide is (b) at leapt 75%, $0%, 85%, 90%, 91%, 93%, 94%, 95%, 90%, 97%, 98%;
or 99% identical to the N9 NA polypeptide Of SEQ. ID lti10:9, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1,12, 13, or all /4 of the following amino acid residues relative to SEQ ID NO:9 When aligned by protocol 1 or protoCol 94P, 95L, 98N, IOOS/A, 126Q1M, 1521', 1581, 160WAIDT, 161V, 191Q, 198Y, 2001, 439S, 441V.
81 The polypeptide of any one of claim 78-80c wherein the polypeptide includes one or more aft. following sets of.4mito. acid residues relative to SEQ. ID NO:9 when aligned by protocol I or prot6e0i 2 96C/159C;
96C/159C/441V;
96A144 IA;
96A1126M/4391/441A;
95L/96A/126M/4391/441A;
126M/4391441A;
951,11126M/4391/441 A
9011.91T;
98N/1 00A; and/or 35 94p/191T/2001.
82. The polypeptide of any one of clairn 78-80, wherein the.pplypeptide includes three or more of the listed amino acid residnes relative to; SEQ NO:9 When aligned by protocol 1 or protocol 2.
$3. The polypeptide of any one of claim 78-80, wherein the polypeptide includes five or more of the listed amino acid residues relative to SEQ ii) NO:9 when aliened by protocol I
or protocol 2.

84. The polypeptide of any one of claim 7840, Wherein the pot pride includes SeVerl or more of the listed amino acid residues relative. to SEQ ID. Na9 when aligned by protocol 1 or protocol 2.
85. The polypeptideof any one of claim 7&80. wherein thepolypeptide includes nine or more of the fisted amino acid residues relative to SgQ fp NO:9 when aligned by protocol or protocol 2.
86, The non-naturally Occurring 'polypeptide of dant' =wherein the Sty-peptide is at 9 Least 75%, 80%, 85%, 90'4, 91%,,M, 93%, 94%, 95%, 96% 97%, 98%, Or 99%
identical to the N9 NA polypeptide of iSEQ ID NO:9, and wherein the non-naturally occurring pOlypeptide includes a 103Q amino acid mutation relative to $EQ 10 NO:9 when aligned by primped 1 or may inch* a combination of 1600/E and 172V arrtinti acid residues relative to SE() ii) NO:9 when aligned by protocol 1 or protocol 2, 87, A composition, comprising one or more Of the polypeptides of any preceding Claim linked tO::a: Scaffold, $8. The composition of Claim 87, wherein the scaffold comprises a protein scaffold.

89. The:compositten of claim -88, wherein the csolypeptide is ova lently linked to a protein subunit of the protein 'scaffold to fttra a fusion protein.
90, A nucleic acid: encoding the polypeptide Of any preceding clan, or the fusion protein of claim 89, 91. An expression vector comprising the nucleic acid of laim 1:.X) operatively lipked to a suitable control sequenet 92. A host:Cell eontPrising the nucleic acid of claim 90, the exPit ssiori Vector of claim 91, and/or the poblvoide of any preceding claim.
93. A pharmaceutical composition, comprising :(a) one or more of the polypeptides, fusion protein, composition, nucleic acid, expression vector, -and/or the host cell of any preceding claim and (b) a nharrnaCentiCally aCceptable carrier, 94. A vaccine comprising:
(a) one or more of the .polypcn tides, fasion protein, composition, nucleic acid, eNpression vector; and/or the host cell of any preceding claim; and (b) a pharmaceutically acceptable carrier.
95, A method for treating or limiting development of an influenza infection, comprising -administering to 0 subject in need thereof an amount effective to WPM or limit OCVOicspg1Ctit Of the influenza: infection of a polypeptide, fusion protein, composition, vaccine, nuclie acid, expression vector, host cell, pharmaceutical t.Toruposition, andlor vaccine of any preceding.
claim.

Claims

Wc claim

1. A non-mturally occurring mutant neuraminidasc (NA). polypeptidc that improves expression and/or modifies the open/closed tetrameric conformational state of the NA
polypeptidc.

2. The non-naturally occurring poiypeptide of daim 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N I NA polypeptide of SEQ ID NO:1, and wherein the non-naturally occurring polypeptide includes I. 2, 3, 4, 5, 6, or all 7 of the following amino acid residues relative to SEQ ID NO:I when aligned by protocol 1 br protocol 2:
16IT/A, 'OSA, 165'171, 1661?, 196Q, 203Y, 444V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the NI NA polypeptidc of SEQ ID NO:1, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, .10, 1I, 12, 13, 14, 15, 16, or all 17 of the following amino acid residues relative to SEQ ID NO:1 when aligned by protocol 1. or protocol 2:
1.61T/AN/S/T, loot, 408M, 419V, 99P, 103N, 105A, 131Q/M, 1631/1., 165'17S/V/A/1, 166P, 17/1, 196Q/T, 203Y, 20544421, 444V.

3. The polypeptide of claim 2, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the NI NA polypeptide of SEQ NO:1, and wherein the non-naturally occwring polypeptide includes 1, 2, 3, 4, 5, 6, or all 7 of the following amino acid residues relative to SEQ 11) NO:1 when aligned by protocol 1 or-protocol 2:
(1) 161T/A, 105A, 157K, 165T11, 166P, I 96Q, 203Y, 444V; or (ii) 105A, 165T/1, 166P, 196Q, 203Y, 444V.

4. The poiypeptide of claim 2, wherein the.pol ypeptide is (15) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N I NA polypeptide of SEQ ID NO:1, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5õ 6, '7, 8., 9, 10, 11. 12, .13, 14, 15, 16, or all 17 of the following amino acid residues relative to SEQ ID NO:1 When aligned by protocol 1 or protocol 2:

:(1) 16ITI.AJWSIT, 1001,, 408M, 419V, .99P, WIN,. 105A, 131Q/M, 1.63111.,, 165TISNIA/1, P. 1771, 196Q/T,.203Y,..205i,4e-121, 4V7, 61-(0 99P, 103N, 105A, 131Q/M, 1631/L, 165I/SAI.INI, 166P, 1771, 196Qfr, 203Y, 2051, 4421, 444V.

5, The polypepticle of. anyone of el a tn.2-4, wilcrein the polypepticte iaci ttles one or plate of the. &flowing setg. anrino acid mskhies tiv o SEQ
ì.1 Nal when aligned by protpcol 1 or prptocol 161V/172A;
100L/498M;

103N/105A;
1141/166P;
101C/164C;
101C/164C/174V;
101C/122V/164C/174V/444V;
122V/444V;
131Q/4421;
101A/163L/444A;
131M/4421;
101A/131M/1631-14421/444A;

131M/163L/4421/444A;
100L/131 1N.N 1 6344421/444A;
99P/.1.00L/161W165S?'172A/1771/196Tq051/408}/419Y;
99P/.100L/161W165S/172A/1771/196174081t/419V;:
99P110011,1131Q/1.61.V1165S/1.72A/1771/196T/205.1/408W419V;
99P/100L/131Q1161W165Sll.72A11771/196T/408M1419V;
991/10011101AlimM1161W1631j165SII72A117711196P20.51i408M419V74421/444A.;
99P/100L/101A/131M/161W163L/165S1.172A/ l 771/ I 96T/408M/419V/4421/444A;
9W.161V/165S/1.72A1177111.967:7105I;
99P/161V/165 S/172A/1771/196T;
99P/131Q/161V/165S/172A/1771/196T/2051;

99P1131Q/1.61\71.65S1172A/1771/196T;
9.9P/101A/131M/1.61V/163L11.65S/1.72All 771/196172051/4421/444A;
99P/101A/131A1 161W1611.1165S/172.A/1771,1961744.21/444A;
99P/196T;
99P/196T/2051;
99P/1771/196T; and/or 99P/1771/196T/2051; or (ii) 103N/105A;
1141/166P;
101C/164C;
101C/164C/174V;
101C/122V/164C/174V/444V;
122V/444V;
131Q/4421;
101A/I1631_1444A;
131M/4421;
101A/131M/163L/4421/444A;
100L/101A/131M/1631_14421/444A;
131M/163L/4421/444A;
100L/1 3 1NIi163L/44211414A;
99P/100L/161 WI 65S/1 7.2A/1. 771/196T/2051/408M141.9V;
99N1001.1131Q/1.61V/165S11.72A11771/196172051.1408M/419V;
99P/100L/131Q/161V/165S/172A/1771/196T/419V;
99PROOL/101A11311\41161V/16314/165S/172A/1771/196T/2051/408M/419V/4421/444A;
99P,!`.1.00L/10141.1.31M/16.1y/1-631,/165S/MA/177111961/408M/419V/4421/444A;
99W1.61 VII 65 S/.172A1.11771/1.96D2051;
99P/161V/165S/I 72Ai'177171.96T;
99W13.1.Q/161V11 65S/172A/1771/ / 96T/2051;
99P/13.1Q/16.1V/1.65S/17.2A/177111 96.1";
.99Pno An 31M1161 V/16311165S7172A11.77/1 96T/2051/4421/444A;
99P1101AMTM/161 fIci31./165S/171A117.714 96T/4421/444A;
99P/196T;
99P/196T/205I;

99P/1771/196T; and/or 99P/17711196T/2051.

6. The polypeptide of any one of claim 2-4, Wherein. the polypeptide includes three or more of the listed amino acid residues relative to SEQ ID NO:1 when aligned hy protocol 1 or protocol 2.

7. The polypeptide of anyone of claim 2-4, wherein the polypeptide includes five or more of the listed arnino acid residues relative -to SEQ. ID Nal when aligned by protocol 1 or protocol 2,

8. The polyp...pride of any .one of claim 2-4, wherein. the polypeptide includes seven or more of the listed arnino acid residues relative to SEQ 113 NO:1 when aligned by protocol 1 or protocol 2.

9. The polypeptide of any one of claim 2-4, wherein the polypeptide includes nine or more of the listed amino acid residues relative to SEQ 1D NO:1 when aligned by protocol 1 or protocol 2.

The polypeptide of any one of claim 2-9, wherein the polypeptide further comprises 1, 2, 3, 4, 5, or all 6 of the following residues relative to SEQ 1.1.) NO:1 when Mimed by protocol 1 or protocol 2: 105S, 106V, 1631, 166V, 210G, 442S.

11. The polypeptide of any one of claim 2-10, wherein the polypeptide includes One or more of the following sets of amino acid residues relative to SEQ113 NO:1 when. aligned by protocol I. or protocol 2:
99P/1001/161V/1.65S/172A/1771/196T/20511408M/419V;
991)1100L1161V/165S/172A/1771/196T/408M/419V:
99P/10C/131Q/161 WI 65S/172A/17711196T/2051/4084/419V;
99P/1001/131Q/161W] 65S/172 A/177111 96T/408M/419V;
99P/100L/101A/131M/161V/163L/165S/172A/1771/196T/2051/408M/419V/4421/444A;
99P/100L/101A/131M/161V/163L/165S1172A/1771/196T/408M/419V/4421/444A;
99P/161V/165S/172A/1771/196T/2051;

99P/161V/165 S/172A11771/196T;
99P/131Q/161V/165S1172A/I 771/196T/2051;
99P/131Q/161V/165S/172A/1771/196T/;
99P/101A/131M/161V/163L/165S/172A/1771/196T/2051/4421/444A; and/or =
99P/101A/131M/161V/163L/165S/172A/1771/196T/4421/444A.

12. The non-naturally occurring polypcptide of any one of elaims 2-11, wherein the polypeptide comprises the amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid selected from the group consisting of NI mutants listed in Table 1 (SEQ ID NO:1 (1-32 and 39-95), when aligned by protocol 1 or protocol 2, wherein the polypeptide includes all of the residues listed in Table 1 for an individual Ni mutant listed in Table 1.

13. The non-naturally Occurring polypeptide of claim 1, wherein the poiypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypeptide of SEQ ID NO:2, and wherein the non-naturally occurring pOlypeptide includes 1, 2, 3, 4, 5, 6, 7, or ail 8 of the following amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2:
low, 1314, 16.2P, 165SIT, 166V; 195Q, 202Y, 443S; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypeptide of SEQ ID NO:2, and wherein the non-naturally occurring polypcptide inchides.2, 3, 4, 5, 6, 7, 11, 9, 10, 1/, or all 12 of the following atnitio acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2:
101CIA, 103N, 105A, 106V, 131M, 162P, 1631, 165S/T/A/1, 166V, 195Q, 202Y, 443S.

14. The polypeptide of claim 13, wherein the polypeptide is (a) at feast 75%, 80%, 85%, 9(r4i, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypeptidc of SEQ ID NO:2, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 1, or all 8 of the followina amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2:
101C, 131M, 162P, 165S/T, 166V, 195Q, 202Y, 443S.

15. The polypeptide of claim .13, wherein the polypeptide is (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N2 NA polypeptide of SEQ 113 NO:2, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 3õ 9, 10, 11, or all 12 of the following amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2:
101C/A., 103N, 105A, 1.06V, 13 I M, 162P, 1631, 165SITIA/1, 166V, 195Q, 202Y, 443S.

16. The polypeptide of any one of claim 13-15, wherein the polypeptide inehules one or more of the following sets of amino acid residues relative to SEQ ID NO:2 When aligned by protocol 1 or protocol 2:
103N/105A;
101C/164C;
101C/164C/173V;

10M/101A/131M andlor l 0011131M/I 631-

17. The polypeptide of any one of claim 13-15, wherein the polypeptide inchides three or more of the listed amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2.

1X. The polypeptide of any one of claim 13-15, wherein the polywptide inehides five or more of the listed amino acid residnes relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2.

19. The polypeptide of any one of claim 13-15, wherein the polypeptide incindes seven or more of the listed militia acid residues relative to SEQ ID NO:2 when aligned by protocol I
or protocol 2.

20. The polypeptidc of any one of claim 13-15, wherein the polypeptide includes nine or more of the listed amino acid residues lelative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2.

21. The non-naturally occurring polypeptide of any one of elaims 13-20, wherein. the polypeptide comprises the amino acid sequence at least 75%; 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid Selected from the group consisting of N2 mntants listed in Table 1 CSEQ ID NO:33-34X when aligned by protocol 1 or protocol 2, wherein the polypeptide includes all of the residues listed in Table 1 for an individual .N2 mutant listed in Table 1.

22. The non-naturally oceuninu polypepticle a claim 1, wherein the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or.99%
identical to the N2 NA polypeptide of SEQ NO:2, and wherein the non-naturally occurring polypeptide includeS a 1654wE amino acid residues relative to SEQ ID NO:2 when aligned by -protocol 1 or protocol -2, or includes 2 or 3 of 165Q/E, 176V, 195S amino acid residues relative to SEQ ID NO:2 when aligned by protocol 1 or protocol 2.

23. The non-naturally occurring polypeptiik of claim 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9'7%, 98%, or 99% identical to the N3 NA polypeptide of SEQ ID NO:3, and wherein the non-naturally occurring pOlypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all I 1 of the following amino acid residu.es relativeto SEQ ID NO:3 when aligned by protocol 1 or protocol 2:
103N, 105S,131Q/M, I 57T, 165.S/1, I 66P, 196Q., 203Y, 2051, 443S, 445V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N3 NA polypeptide of SEQ ID NO:3, and wherein the non-naturally occurring polypeptide includes 2, 3,-4, 5., 6, 7, 8, 9, In, 11, 12, or all 13 of the following amino acid residues relative to SEQ 1D N011 when aligned by protocol 1 or protocol 2:
103N, 105S, 106V, 131Q1M, 157T, 1631.õ 165SIIIVIA, 166PIV, I96Q, 203Y, 2051, 443S, 445V.

24.. The polypeptide of claim 23, *herein the polypeptide is (a) at feast 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N3 NA polypeptidc of SEQ TD NO:3, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the followinn amino acid residues relative to SEQ ID NO:3 when aligned by protocol 1 or protocol 2:
103N, 105S, 13 IQ/M, 57T, 165S/1, I 66P, 196Q, 203Y, 2051, 443S, 445V.

25. The polypeptide of claim 23, wherein the polypeptide is (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N3 NA polypeptide of SEQ 113 NO:3, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8õ 9, 10, 11, 12, or all 13 attic following amino acid residues relative to SEQ ID NO:3 when aligned by protocol 1 or protocol 2:
103N, 105S, 106V, 13IQ/M, 157T, 163L, 165S/I/V/A, 166P/V, 196Q, 203Y 2051õ
443S, 445V.

26. The polypeptide of arty one of claim 23-25, wherein the polypeptide includes one or more of the following sets &amino acid residues relative to SEQ. ID NO:3 when aligned by protocol 1 or protocol-2:
101C/164C;
101C/164C/174V;
101C/164C/174V/445V;
101A/445V;
101A/131M/445A;
131M/445A;
1141/166P;
101A/163L/445Võ;
101A/13 I'M/16314445A; and/or 131M/16311445A.

27. The polypeptide of any one of claim 23-25, wherein the polypeptide includes three or more of the listed amino acid residires relative to SEQ. ID NO:3 When aligned by protocol 1 or protocol 2.

28.. The polypeptide of any one of claim 23-25, wherein the polypeptide includes five or more of the listed arnino acid residues relative to SEQ ID NO:3 when aligned hy protocol or protocol 2.

29. The polypeptide of any one of claim 23-25, wherein the polypeptide includes.seven or more of the listed amino acid residues relative to SEQ ID .NO:3 when aliened by protocol 1 or protocol 2.

30. The polypeptide of any one of claim 23-25, wherein the polypeptide inchides nine or more of the listed amino acid residues relative to SEQ ID NO:3 when aligned by protocol 1 or protocol 2.

31. The non-naturally occurring polypeptide of claim I, wherein the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N3 NA polypeptide of SEQ ID NO:3, and wherein the :non-naturally occurring polypeptide includes 1, .or both of the following amino acid residues relative to SEQ ID NO:3 when aligned by protocol 1 or protocol. 2: 196S, 165Q/E.

32. The non-naturally occurring polypeptide of claim 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N4 NA polypeptide of SEQ ID NO:4, and wherein the non-natnrally occurring polypcptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I 1, 12, 13, 14, 15, or all 16 of the following amino acid residues relative to SEQ ID NO:4 When aligned by protocol 1 or protocol 2:
160V/S/T/A, 409M, 102N, 104S/A, 105V, I12D, 130Q/M, 162L, 164S/T/A/I, 165P, 176I, 195Q, 202Y, 204J, 443I, 445V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N4 NA polypeptide of SEQ ID NO:4, and wherein the non-naturally oecurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or all 16 of the following amino acid residUes relative to SEQ ID NO:4 when aligned by protocol 1 or protocol 2:
160V/S/T/A, 409M 102N, 104S/A, 105V, 112D, 130QM, 162L, 164S/T/A/I, 165P/V, 176I, 195Q, 202Y, 204I, 443I, 445V.

33.. The polypeptide of claiin 32, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N4 NA polypeptide of SEQ ID NO:4, and wherein the non-naturally oecuiring polypepfide includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following amino acid residues relative to SEQ ID NO:4 when aliened by protocol 1 or protocol 2:
(i) 160V/S/T/A, 489M, 102N, 104S/A, 105V, 112D, 130Q/M, 162L, 164S/T/A/I, 165P, 176I, 195Q, 202Y, 204I, 443I, 445V; or :(1i) 102N, .104S/A, 1:05V, 112D, 130Q.:M, 162L, 164S/T/A4, 165P, 1761, 195Q, 202Y,.2041, 4431, .445V.

34 no polypeptide oFciaim 3.2. Avbeteitt.the pO1ypeptidc.is :(b.). at. least 75.4.,.80%,..85%, 90 A.91%, .92%, 93%., 94%, 95%, 96%i, 97%; .98%, or. 99% jicicntical, to the N4 NA polypeptide. SEQ ID N.Q.:4, and wherein the notk-naturally c!ceurringpoly)eptidc. includes 2, 3, 4, 5õ. 6,. 7, 9õ. IL 12, 13,14, 15, or all 10 ofthe f011.o*ing amino acid rcstducs relanrelp SEQ1D N9.4. *ben aligned 'by wiatocol. 1 or prOtoccil 2;
160V/SITIA, 4091,102N,104S/A, 105V, 112D, 130Q/11.1, 162L, 164S/T/A/I, 165PN, 1761, 195Q, 202X..2041,44.31, 445V; pc (ii) 10.2N, 104 VA, 105V, 1.12, .130Q/M, 1621, 164S/T/AIL 1.65PN, 1761, 195Q, 2021i',. 2041, 4431õ 445V.

35, The polyp** cf any one .of dairti 32-34, u4tereitt the polypeptide.
includes one or .ttioroof the tonowing. sagOfatnitvPacid residne5 relative to SEQ ID
NO:41iihen atianed by prOtoed .1 Or. prOtOeol .2:
(1) 160V/SITIA;
160V/171A;
102N/104A;
100C/163C;
100C/163C/173V;
100C/163C/173V/445V;
130Q/4431;
100A/162L/445A;
13W/4431;
1.00A11301%11162L/4431/445k 1.30W1621,144311445A;
1761/2041;
100C112.1V1163C/173V/445V; andietr 121 V/445V.;:or 1 02N/104A;

100C/1.63C;
10001.63C/173V;
100C/163C1173V/445V;
130Q/4431;
100A/162L/445V;
130M/4431;
100A/130M/162L/4431/445A;
130M/162L/4431/445A;
1761/2041;
100C/121V/163C/173V/445V; and/or 121V/445V.

36. The polypeptide of any one of claim 32-34, wherein the polypeptide includes three or more of the listed amino acid residues relative to SEQ ID NO:4 when aligned by protocol I
or protocol 2.

37. The polypeptide of any one of claim 32-34, wherein the polypeptide includes five or more of the listed ainino acid residues relative to SEQ 11) NO:4 when aligned by protocol 1 or protocol 2.

3. The polypepticle of any one of claim 32-34, wherein the polypeptide includes seven or more of the listed amino acid resichtes relative to SEQ. ID NO:4 when aligned by protocol 1 or protocol 2.

39. The polypeptide of any one of claim 32-34, wherein the polypeptide includes nine or more of the listed arnino acid residues relative to SEQ 1D NO:4 when aligned by protocol 1 or protocol 2.

40. The non-naturally oceurrinu polypeptide of claim 1, wherein the piatypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to thels14 NA polypeptide of SEQID NO:4, and wherein the non-naturally occurring polypeptide includes I, or both of the following ainino acid residues relative to SEQ11) NO:4 when aligned by protocol 1 or protocol 2: 1640.1E, I 95S.

41. The non-natural1y occurring polypeptidc of claini 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ ID NO:5, and wherein the non-naturally occurring pOlypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or al/
13 of the following arnino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2:
971,, 410M, 96P, 98A, 100N, 102SIA, 1 IOD, 128QM, 1601, 162V/A/1, 163V/P, 193Q/T, 4451; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ ID NO:5, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 3, 9, 10, II, 12, 13, 14, 15, or till 16 of the following amino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2:
97L, 41.0M, 96P, 9$A, 100Nõ 102S/A, 103V, 110D, 128Q/M, 154T, 1601, 162V/A/I/T, I 63V/P, 1741, 193Qrr, 4451.

42. The polypeptide of c1aim41, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%õ 96%, 97%, 98%, or 99% identical to-the N5 .N A polypeptide of SEQ 113 NO:5, and wherein the non-naturally occurring polypeptide includes 1õ 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13' of the following arnino acid residues relative to SEQ ID NO:5 when aligned hy protocol 1 or protocol 2:
(i) 971, 410M, 96P, 98A, 100N, 102S/A, 110D, 128Q1M, 1601, 162V/A/I, 163V/P, 93Q1T, 4451; or (ii) 96P, 98A, WON, 102S1A, 110D, 128Q/M, 1601, 162V/A/1, 163V/P, 193QTT, 4451.

43.. The polypeptide of claim 41, *herein the polypeptide is (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N5 NA polypeptide of SEQ ID NO:5, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following arni:no acid residues relative:to SEQ ID NO:5 when aligned by protocol 1 or protocol 2:
(i) 91L, 410M, 96P, 98A, MON, 102S/A, 103V, 110D, 128011, 154T, 1601, 162V/A/I/T, 163V1P, 1741, I93QIT, 4451; or 00 9op, 98k 00N,102S/A, 1.03V, 110D, 128Q/M, 154T, 1601, 162V/A/I/T, ifiWIP, 1741, 193QT, 4451,

44: no polypeptide of arty. one:of claim4E43. herei e polypepcide íe.des ofte. or more of the foll6wingsets'of arnito.acid reidues: relative to SEQ. la NO.:5 vkihen .aligned by protocol l or protocol 2:
(i) 97L/410M;
100N/102A;
98C/161C;
12SQ/445I;
128M/4451;
9.8A/ 128W4451447A;
97L/9-$A1128M4401/447A;
1.2814I4451,447k C76P/193T;.
1 111/163P;
90/.19:317/202:1;
96P1741/1 93T; mdfor 96P/1741/1931/2021; or (ii) 100N/102A;
98C/161C;
128Q/4451;
128M/4451;
98A/128M/4451/447A;
971198A11281/44511447A;
281W4451.1447k 971)120$114451/447A;
96P/193T;
111I/163P;
96P/193T/2021;
96P/174I/193T; and/or 96P/1741/193T/2021.

45. The polypeptide of any caw of claim 41-43, wherein the polypeptide includes three or more of the listed amino acid residUes relative to SEQ ID NO:5 when aIiimed by protocol or protocol 2.

46. The polypeptide of any one of claim 41-43, wherein the polypeptide includes five or more of the listed amino acid residues relative to SEQ. ID NO:5 whc:n aligned by protocol 1 or protocol 2.

47. The polypeptide of any one of claim 41-43, wherein the polypeptide includes seven or more of the listed amino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2,

48. The palypeptide of any one of claim 41-43, wherein the polypeptide inchides nine or tnore of the listed amino acid residues relative to SEQ ID NO:5 when aligned by protocol 1 or protocol 2.

49. The non-naturally occurring polypeptide of elairn 1., wherein the polypeptide is at least 75%, 80%õ 85%, 9.4)%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N5 NA polypeptide of SEQ ID NO:5, and wherein the non-naturally occurring polypepticle includes 1, or both of the following amino acid residues relative to SEQ. ID NO:5 when aligned by protocol. .1 or protooal 2: 162 Q/13, 200S.

50. The non7naturally occurring polypeptidc of claim 1, wherein the polypepticle is (a) at least 75%, 80%, 85%, %Wu, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N6 NA polypeptidc of SEQ ID NO:6, and wherein the non-naturally occturing polypeptidc includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 1 I of the following amino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2:
99P, 103N, 113D, 13.IQ, 1611, 162P, 16315 165SITNIA, 196Q, 203Y, 445S; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N6 NA polypeptide of SEQ ID NO:6, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5,6, 7õ 8, 9, 10, I I, 12,13, 14, 15, or all 16 of the following:amino aeid tesidues relative tO SEQ ID NO:6 When aligned by protoccil 1 or protocol 2:
99P, 103N, 1055, 106V, 1.131, 131Q, 157T, 1611, 162P, 16311, 165S/TNIAII, 1.66WP, 196Q, 203Y, 445S.

51. The pplypeptide =of cIOn 50, yherOp the polypeptickis (tt) at lea:* 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,, 95%, 96%, 97%, 98%, or 99% ideOtical to 0101%16 polypeptide OfSEQ.10 NO:6, and =Aerein the mart-rtaturally occitrring i)olypeptide includes Iõ 2, 3, 4, 5, 6.; 7, 8, 9; 10, or ali 11 of the followiug LLO
acid re$iduts rciattre to SEQ ID NO:6. when aligucd by prOtoco1 I Or pi-OW:col 103N, 11 3I), 131Q, 1611:õ1t52, 1631, OWTNIA, 190% 203Y, 445S.

52. The polypentide of elaiin50, Wherein the polypotide i5 (.l)) atIca5k 75%, 80%, 85%, 9", 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ideOtieal to the N6 NA po.13ipeptide of SEQ 11.NO:6, and *herein the attualIy occurring polypeptide itiektdes 2, 3, 4, 5, 6, 7, 3, 9, 10. 11, 12 13, 14, or all 15 of the following amitio aeid residites relative o SEQ 11) NO:6 Nixeri tt1itned by piOtoco1 1 Of protocol 2:
99P, 103N, 105S, 106V, 113D, 131Q, 157T, 1611, 162P, 1631/L, 165S/T/V/A/I, 166V/P, 196Q, 203Y, 445S.

53, I*
polypoptide of atly ottc of claim 50-52, :wherein the pOiyp4ticle iocludes one or erkwe of the followirqt'sets of amino :acid residnes: reintiVe t SEQ ID NO:6 NVIlen aligned by protocol 1 or protocol 2:
101C/164C, 101C/164C/174V;
101C/164C/174V/447V;
122V1447V;
101A/163L;
99P/196T; arKl!or 99P/196T/2051.

54. The polypeptide of any one of claim 50-52, Wherein the polypeptide inchides three or more of the Iisted atnino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2.

55. The polypeptide of any one of claim 50-52, whereM the polypeptide includes five or more of the listed aminciacid residues relative to SEQ ID NO:6 when aligned by protocol or protocol 2.

56. The polypeptide of any one of claim 50-52, wherein the polypeptide includes secien. or more of the listed amino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2.

57. The polypeptide of any one of claim 50-52, wherein the polypeptide includes nine or more of the fisted amino acid residues relative to SEQ ID NO:6 when aligned by protocol 1 or protocol 2.

58. The non-naturally occurring polypeptick of claim 1, wherein the polypeptide is at least 75%õ 80%,. 85%, 90%, 91%, 92%, 93%õ 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N6 NA polypeptide-of SEQ ID NO:6, and wherein the non-naturally occurring polypeptide includes a 165E amino acid mutation relative to SEQ ID NO:6 when aligned by protocol 1, arid optionally also includes a 177V amino acid mutation relative to SEQ ID NO:6 when aligned by prOtocol 1 or protocol 2.

59. The non-naturally occurring polypeptide of elann 1, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%., or 99% identical to the N7 NA polypeptide of SEQ ID NO:7, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 1.1 of the following amino acid residues relative to SEQ ft) NO:7 when aligned by protocol I 'or protocol 2:
98P, 102N, I04S, 1120, 130Q, 156T, 1621, 164V/A/I, 165V, 202Y, 448V; or (b.) at least 7.5%i 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the -N7 NA polypeptide of SEQ ID NO:7, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 of the following amino acid residues relative to .SEQ ID NO:7 when aligned by protocol 1 or protocel 2:
98P, 102N, 1045, 112D, 130Q, I 56T, 1621, 164V/AII, I65V, 1761, 202Y, 4461, 448V.

60. The polypeptide of claim 59, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%; 96%, 97%, 98%, or 99% identical to the N7 NA polypeptide of SEQ ID NO:7, and wherein the non-naturally occurring polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the following arninb acid residues relative to SEQ 113 NO:7 when aligned by protocol 1 or protocol 2:
9$P, 102N, 104S, 112D, 130Q, 156T, 1621, 164V/A/I, 165V, 202Y, 448V.

61. The polypeptide of claim 59, Wherein the polypeptide is (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N7 NA polypeptide of SEQ ID NO:7., and wherein the non-natuntlly occturing polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, .12, or all 13 of the following amino acid residues.relative to SEQ NO:7 when aligned by protocol 1 or protocol 2:
98P, 102N. 104S, 112D, 130Q, 156T, 1621, 164V/A11, 165V, 1761, 202Y, 4461, 448V.

62. The polypeptide of any one of claim 59-61, wherein the polypeptide includes one or more of the following sets of amino acid residues relative to SEQ ID NO:7 when aliened by protocol 1 or protoCol 2:
100C/163C;
100C/163C/173V;
100C/163C/173V/448V:
991_1446T/448A;
98P/195T;
130Q/4461;
446I/448A;
98P/195T/2041;
98P/1.761/1.95T; andior 98P/1761/1.95T/2041.

63. The polypepticle of any one of claim 59-61, wherein the polypeptide includes three or more of the listed antino acid residues lelative to SEQ ID NO:7 when aligned by protocol 1 or protocol 2.

64. The polypeptide of any one of claim 59-61, Wherein the poly-peptide includes five or more of the listed amino acid residues relative to SEQ :ID NO:7 when aligned by 'protocol 1 or protocol 2.

65. The polypeptide of any one of claim 59-61, whereM the polypeptide includes seven or more of the listed amincracid residues relative to SEQ ID NO:7 when al igned by protocol I
or protocol 2.

66. The polypeptide of any one of claim 59-61, wherein the polypeptide includes nine or more of the listed amino acid residues relative to SEQ ID NO:7 when aligned by protocol I
or protocol 2.

67. The non-naturally occurring polypeptiik of claim 1, wherein the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N7 NA polypeptide of SEQ. ID NO:7, and wherein the non-naturally occurring polypoptide includes one or both of the following amino acid mutation relative to SEQ 11) NO:7 when aligned by protocol t or protocol 2: 164Q/E, 195S.

68. The non-naturally occurring polypeptide of claim 1., wherein the polypeptide is (a) at least 75%, 80.%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally occurring polypeptide inchides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the following ainino acid residues relative to SEQ ID NO:8: when aligned by protocol 1 or protocol 2:
408M, 101N, 103kS, 11113, I29Q, !am, 163SfriVIAil, 164V, 194Q, 201Y, 4421; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA polypeptide of SEQ. ID NO:8, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 1.1, .12, 13, 14, or al1.15 of the following amino acid residues relative to SEQ ED NO:8 When aligned by protocol 1 or protocol 2:
981õ 40810., .101N, 101A/S, 104V, 1110, 129Q/14, 161P/1õ 1635.11WANSIT, 1 64V/P, 1751, 194Q, 20 I Y, 2031, 4421

69. The polypeptide of cloim 68, witerein. the poinieptide is (a) at least 75%, litft, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tO the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally occurring pOlypeptide includes 1, 2, 3, 4, .5, 6, 7, S. 9, 10, or all 11 of the following amino acid residues:relative to SEQ ID Na8 when aligned by protocol 1 or protocol 1 (i) 40MI, 101N, 10.3A5, 111D, 129Q, 160P, I 61L, 163S/TIVIAll, I64:V, 194Q, 201Y, 442i or OD 101N, 103A/S, 11 D, I 29Q, 169P, 161L, 163S/T/V/A/I, 164V, 194Q, 201Y, 4421.

70. The polypeptide of claim 6k whigein the polypeptide is (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N8 NA riolypeptide ofSEQ ID NOKI.., And ,,s71-terein the bon-naturally peCuttitig, OlYpeptide itIcludes 2, 3, 4, 5, 6, 7, 8,9, 1(1, 11, 12, 13, 14, or all 15 of the fp-Ho-Wing arnink) acid residues relative to:SEQ ID Nati When alignedb7:;, protocol 1 or tstOcol 2:
(i) 9S1, 408M, 101N, 103A/S, 104V, 111D, 129Q/M, 160P, 161L, 163STriVINIIVL I 64V/P 1751, I 9-1Q., 201Y, 2031, 4421, or (1) I MN, 103A.,s, IQ4V, 1110, I 29Q/K 100e, 1611, 163S/TN/A.Ii:Sa, 164V/P.; 1751, 194Q, 201y, 2031., 4421

71. The polypeoide of any one of Claim 68-70, :wherein the pOiypepticie includes Qne or

PIM of the following:=sets=of amino residnes: relatiVe to SEQ. ID Na8 when aligned by protocol 1 or protocol 2:
(i) 98L/408M, 160P/163S;
101N/103A::
99C/162C;
99C/162C/172V;
129Q/4421;
99A/16 IL;
99A/161L/4421;
161L/4421;

129M/442t 99A/129M/161114421/444A;
981,199A/129M/1151114421/444A
129M/161 IS442114.114A;
98L/129M1161114421/444A; artdior 1751/2031; or (ii) 160P/163S;
101N/103A;
99C/162C;
99C/162C/172V;
129Q/442I;
99A/161L;
99A/161L/4421;
161114421;

99A/129M/161L/4421/444A;
98L199A/129M/16 L/4421/444A;
1 294(1611.14421/144A;
98V129M11611-44214144A; touLtot 175112031.
=72. The polypeptick of any csne of claim 68-70, wherein the pO13ipdpti3c *Judea three ot trio* ofthc Wed atninO acid residues relative to SEQ ID NO:8 'when allotted by prOtoc61 1 or protocol 7,

73, The polypeptide of arty pm of clant 6&-70,hre*polypeptidc includes five ot rc of the fisted annno Ezeld regidaOg rdatiVe tO SEQ11) NO:8 haligtd by protocol 1 or protocol 2.

74.= The polypeinide of any -one of claim 68-70, wIlerein the polypeptide includes geven or Dim of the listed annoo ncid residues reigive to SEQ 11.:).NO:8. when nlioned by protocol or protocol 2.

75. The polypeptide of any one of claim 68-70, Wherein the polypeptide inchides nine or more of the listed amino acid residues relative to SEQ ID NO:8 when aligned by protocol 1 or protocol 2.

76. The non-naturally occurring polypeptide of any one of claims 80-85, wherein the polypeptide eomprises the amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9$%, or 99% identical to the amino acid selected from the group consisting of N8 mutants listed in Table 1 (S.EQ ID NO:35-38), when aligned by protocol 1, wherein the polypeptide includes aII attic residues listed in Table 1 for an individual N8 mutant liked in Table I.

77. The non-naturally occurring polypepti& of claim 1, wherein the polypeptide is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the N8 NA polypeptide of SEQ ID NO:8, and wherein the non-naturally occurring polypeptide includes a 163E amino acid mutation relative to SEQ i.D NO:8 when aligned by protocol I , and further may optionally include a I 94S mutation relative to SEQ ID NO:8.
when aligned by protocol 1 or protocol 2.

78. The non-naturally occurring polypeptide of claim 1., wherein the polypeptide is (a) at least 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99'!4) identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide inchides 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 1.0 of the following ainino acid residues relative to SEQ ID NO:9 when aligned by protocol 1 or protoco12:
94P, 951., 100S, 1.26Q/M, 160V/k1, 161V, 191Q, 198Y, 439S, 441V; or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, II , 12, 13, or all 14 of the following amino acid residues. relative to SEQ ID NO:9 when aligned by protocol 1 or protocel 2:
94P, 951õ, 98N, 100SIA, 126Q/M, 152T, 1581, 160V/ANT, 16IV, 191Q, .198Y, 2001, 43.9S, 441V.

79. The polypeptide of claim 78, wherein the polypeptide is (a) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ. ID NO:9, and wherein the non-naturally occurring polypeptide includes 1, 2, 3õ.4, 5, -05, 7, 8, 9, or all 1.0 of the following atnino aCid residues relative to SEQ11) NO:9 when aliened by protocol 1 or protocol 2:
94P, 951., 100S, 126Q/M, 160V/All, 161V, 191Q, I98Y, 439S, 441V.

80. The polypeptide of claim 78, wherein the polypeptide is (b) at least 73%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the N9 NA polypeptide of SEQ ID NO:9, and wherein the non-naturally occurring polypeptidc includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, or all .14 of thc following amino acid residueS.relative to :SEQ ID NO:9 w.hen aligned by protocol 1 or protocol 2:
94P, 951, 98N, 100S/A, 126Q/K 152T, 1581, 160ViAillT, 161V, 191Q, 198Y, 2001, 439S, 441V.

81. The polypepticle of any one of claim 78-80, wherein the polypeptide includes one or more of the following sets of amino acid residues relative to SEQ 113 NO:9 when aligned by protocol 1 or protocol 2:
96C/159C;
96C/159C/441V;
96A/44IA;
96A/126M/4391/44IA;
95L/96A/126M/4391/441A;
126M/4391/441A:
951./126M/4391/441 A;
94P/191T;
98N/100A; and/or 94P/ I 91T/2001.

82. The polypeptide of any one of claim 78-80, wherein the polypeptide includes three or more of the listed amino acid residues relative to SEQ ID NO:9 when aligned by protocol or protocol 2.

83. The polypeptide of any one of claim 78-/0, wherein the polypeptide includes five or more of the listed amino acid residues relative to SEQ ID NO:9 when alioned by protocol 1 or protocol 2.

84. The polypeptide of any one of claim 78-80, Wherein the polypeptide inchidcs seven. or more of the listed atnino acid residues relative to SEQ ID NO:9 when aligned by protocol 1 or protocol 2.

85. The polypeptide of any one of daim 78430, whereM the polypeptide includes nine or more of the lista aminaacid residues relative to SEQ ID NO:9 when aligned hy protocol or protocol 2.

86. The non-qtaturally occurring polypeptide of claim I, wherein the polypeptide :is at least 75%, 80%, 85%, 90%, 91%,:92%, 93%, 94%, 95%, 9.6%, 97%, 98%, or 99%
identical to the N9 NA polypeptide of SEQ 0) NO:9, and wherein the non-naturally occurring polypeptide includes a 160Q annno acid mutation relative to SEQ ID NO:9 when alinned by protocol I , or may inclUde a combination of I 60Q/E and 172V amino acid residues relativeto SEQ ID NO:9 when aligned by protocol I or protocol 2.

87. A composition, comprising:one or more of the polypeptides of any preceding claim linked to a scaffold.

88. The composition of claim 87, wherein the scaffold comprises a protein scaffold.

89. The composition of claim 88, wherein the polypeptide is covalently linked to a protein subunit of the protein scaffold to fi,mn a fusion protein.

90. A nucleic acid encoding the polypeptide of any precedinc.s clann, or the fusion protein of elaim 89.

91.. An expression vector comprising the nucleic acid of claim 90 operativelylinked to a suitable control segue-nee.

92. A host cell comprising the nucleic acid of claim 90, the expression vector of claim 91, and/or the polypeptide of any preceding claim.

93. A pharmaceutical composition, comprising (a) one or rnorc .of the polypeptides, fusion protein, cornpoSition, nucleic acid, expression vector, andlor the host cell of any preceding claim; and (b) a. pharmaceutically acceptable carrier.

94. A vaccine comprising (a) one or more of the polypeptides, aision protein, composition, nucleic acid, expression vector, and/or the host cell of any preceding clairn; and (b) a pharmaceutically acceptable carrier.

95. A method for treating. or limiting development of an influenza infection, comprising administering to a subject in need thereof an amount eff4.1ctive to u-ent or limit development of the influenza infection of a polypeptide, fusion protein, composition, vaccine, nucleic acid, expression vector, host cell, pharmaceutical composition, and/or vaccine of any preceding claim.